A fermentation culture method for increasing the production of Beauveria bassiana

Abstract

This invention provides a method for increasing the fermentation yield of beauveria bassiana, using a basal fermentation medium composed of 50-100 g / L carbon source, 10-30 g / L nitrogen source, and 5-25 g / L inorganic salts. During the 0-120 h fermentation period, a certain amount of L-phenylalanine and valine are added to the basal fermentation medium or during the fermentation process. This method can significantly increase the yield of beauveria bassiana, achieving a fermentation level of over 2900 µg / ml. Furthermore, the process is simple, stable, and suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation, and specifically relates to a fermentation and cultivation method for beautifier. Background Technology

[0002] With the rise of the global trend of sustainable development, green production or green manufacturing has become an inevitable trend for future industrial development.

[0003] Beauvericin (BEA), as a green microbial pesticide, possesses advantages such as high specificity, no harm to non-target organisms, no pollution, low likelihood of inducing resistance, and environmental compatibility. It is widely used as a fungicide, herbicide, and insecticide in large-scale forests and small lawns and gardens. Furthermore, beauvericin exhibits numerous biological activities, including antibacterial, antiviral, and antitumor effects. Its potential applications in treating fatal diseases such as cancer, viral and bacterial infections are increasingly recognized in human medicine, indicating a broad market prospect.

[0004] Beauveria bassiana extract was first isolated from Beauveria bassiana as a secondary metabolite of entomopathogenic fungi. It is a six-membered cyclic tripeptide compound whose molecular structure contains three N-methylphenylalanine and (D)-α-hydroxyisovaleric acid molecules linked by interlacing peptide and ester bonds. Its molecular formula is C1. 45 H 57 N3O9 has a molecular weight of 786.

[0005] Most of the beauveria bassiana produced so far are produced by the genera Beauveria bassiana and Fusarium. However, reports on the production of beauveria bassiana by Fusarium mostly use solid-state fermentation. However, solid-state fermentation has the following drawbacks: (1) It is complicated to operate and has a long fermentation cycle. For example, the solid-state fermentation cycle disclosed in CN105087726A is as long as 20 days; (2) It is difficult to control the evaporation of water in the culture medium, which results in large fluctuations in yield; (3) It is difficult to mix the substrate and metabolites cannot be detected in real time, which is not convenient for the automatic control operation in future large-scale production. Fermentation levels under liquid culture conditions are generally low. The liquid fermentation unit of beauvericin reported by China Agricultural University in 2008, "Enhance dbeauvericin production with in situ adsorption in mycelial liquid culture of Fusarium redolens Dzf2," was 400.24 µg / ml. Li Wei's 2012 paper, "Liquid Deep Fermentation and Regulation of the Antifungal Drug Beauvericin," also showed a liquid fermentation unit of only 488.24 µg / ml in a 50L fermenter. Patent CN.2013107251576 discloses a fermentation unit of beauvericin of approximately 400 µg / ml. Sun Anhui's 2016 study, "Study on Heterologous Expression of Beauvericin in Aspergillus nidus," achieved a maximum beauvericin yield of 668.97 µg / ml through heterologous expression of beauvericin synthase and α-ketoisovalerate reductase in Aspergillus nidus.

[0006] To date, there have been no major breakthroughs reported in the fermentation of beauveria bassiana, therefore there is an urgent need for a high-yield fermentation medium and fermentation method to meet the needs of industrial production. Summary of the Invention

[0007] The purpose of this invention is to provide a fermentation culture method for beauverin, which can significantly increase the fermentation unit of beauverin, and is simple, feasible, and suitable for industrialization.

[0008] The objective of this invention is achieved through the following technical solution: A fermentation culture method for beautifier: Fertile Fusarium fusarium proliferatumThe strain is cultured to obtain a seed culture, which is then inoculated into a basal fermentation medium for liquid fermentation. The fermentation cycle is 7 days. During the first 0-120 hours of fermentation, 1-9 g / L of L-phenylalanine and 0.8-7 g / L of valine are added to the basal fermentation medium or fermentation broth. The basal fermentation medium is the portion of the medium that does not contain L-phenylalanine and valine. The basal fermentation medium contains: 50-100 g / L of carbon source, 10-30 g / L of nitrogen source, and 5-25 g / L of inorganic salts; the carbon source is glucose, maltodextrin, or a combination thereof; the nitrogen source is peptone; and the inorganic salts include potassium dihydrogen phosphate, sodium nitrate, and calcium carbonate.

[0009] The composition of the basic fermentation medium can be: glucose 40-70 g / L, maltodextrin 10-30 g / L, peptone 10-30 g / L, potassium dihydrogen phosphate 5-15 g / L, sodium nitrate 1-3 g / L, and calcium carbonate 1-4 g / L. Preferably, it is: glucose 50 g / L, maltodextrin 25 g / L, peptone 20 g / L, potassium dihydrogen phosphate 10 g / L, sodium nitrate 2 g / L, and calcium carbonate 3 g / L.

[0010] Preferably, 2-6 g / L of L-phenylalanine and 2-5 g / L of valine are added, more preferably 3-4 g / L of L-phenylalanine and 2-4 g / L of valine are added.

[0011] L-phenylalanine and valine can be added directly to the basic fermentation medium, as in the shake-flask fermentation medium of Examples 1-7. Alternatively, L-phenylalanine and valine can be added to the fermentation broth after 24-120 hours of fermentation.

[0012] The addition of L-phenylalanine and valine after 24-120 hours of fermentation can be done in a single addition, as in Examples 8-11, where L-phenylalanine and valine are added to the shake-flask fermentation medium after a certain period of cultivation. Preferably, L-phenylalanine and valine are added to the shake-flask fermentation medium in a single addition within 24-72 hours of cultivation. Alternatively, L-phenylalanine and valine can be continuously added starting after 24 hours of fermentation, as in Examples 12-17, where L-phenylalanine and valine are continuously added to the fermenter medium after 24 hours of fermentation. The duration of supplementation can be 24-96 hours; preferably, the supplementation duration is 96 hours, at which time the supplementation rate of L-phenylalanine is 0.01 g / L·h-0.09 g / L·h, and the supplementation rate of valine is 0.009 g / L·h-0.07 g / L·h; preferably, the supplementation rate of L-phenylalanine is 0.035 g / L·h-0.055 g / L·h, and the supplementation rate of valine is 0.03 g / L·h-0.055 g / L·h.

[0013] The fermentation culture method for beautifier in this invention can be as follows: fermenting fertile Fusarium... fusarium proliferatum The strain was cultured in slant culture, shake flask culture and seed tank culture to obtain seed liquid in the seed tank. The seed liquid in the seed tank was then inoculated into the basic fermentation medium for fermentation tank culture.

[0014] The specific fermentation and cultivation process can be as follows: The seed tank cultivation conditions are as follows: During the cultivation process, the cultivation temperature is controlled at 28.0±0.5℃, the tank pressure at 0.045-0.065MPa, the aeration rate at 0.5-1.0VVM, the stirring speed at 200-550RPM, and the dissolved oxygen at 20% or higher. The seed liquid is obtained after 26 hours of cultivation. The fermentation tank cultivation conditions are as follows: During the fermentation process, the fermentation temperature is controlled at 28.0±0.5℃, the tank pressure at 0.045-0.065MPa, the aeration rate at 0.5-1.0VVM, the stirring speed at 200-550RPM, the dissolved oxygen at 20% or higher, and the total sugar content at 0.5-2.0%.

[0015] The fertile Fusarium proliferatum mentioned in this invention can be fertile Fusarium LF601, with accession number CGMCC 3.1777.

[0016] The beneficial effects of this invention are mainly reflected in: This invention significantly promotes the biosynthesis of secondary metabolites by optimizing the composition and ratio of carbon and nitrogen sources and inorganic salts in the fermentation basal medium, supplementing L-phenylalanine and valine during fermentation, and coordinating fermentation process regulation. This results in a substantial increase in the yield of beauveria bassiana. The invention features a simple operating process, few impurities, stable process control, and is suitable for industrial production, facilitating its widespread application. Detailed Implementation

[0017] The technical solutions in the embodiments of the present invention are described below. The described embodiments are merely a part of the present invention. The following embodiments are used to illustrate the present invention, but should not be used to limit the scope of the present invention.

[0018] In this invention, there are no special restrictions on the materials used; any material source well known in the art may be used.

[0019] In this invention, the beauveria bassiana producing bacterium is a fertile Fusarium (Fusarium wiltii). fusarium proliferatum LF601.

[0020] The strain used in this invention is fertile Fusarium ( fusarium proliferatum LF601 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC 3.1777.

[0021] In this invention, beauveria bassiana can be detected by high-performance liquid chromatography (HPLC) under the following conditions: Chromatographic column: Agilent C18 column (4.6 mm × 250 mm, 5 μm); Mobile phase: Acetonitrile:water = 8:2; Flow rate: 1.0 mL / min; Column temperature: 31℃; Detection wavelength: 254nm; Injection volume: 10 μl.

[0022] I. Beauveria bassiana shake-flask fermentation of basal medium supplemented with L-phenylalanine and valine: Examples 1-7

[0023] (1) Slant culture of strain: After thawing the glycerol tube of fertile Fusarium LF601, it was inoculated into slant culture medium and cultured statically at 28.0℃ for 8 days to obtain slant strain.

[0024] The slant culture medium is potato dextrose agar medium.

[0025] (2) Shake flask culture of strains: Take the slant strains prepared above, inoculate them into shake flask seed culture medium, and culture at 28.0℃ for 72h to obtain shake flask seed liquid.

[0026] The shake flask seed culture medium consisted of: 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, and natural pH.

[0027] (3) Inoculate the above shake flask seed liquid into the shake flask fermentation medium at a volume ratio of 10%, and shake the flask at a speed of 220 RPM.

[0028] The basic fermentation medium consisted of: 50 g / L glucose, 25 g / L maltodextrin, 20 g / L peptone, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, and 3 g / L calcium carbonate, with a natural pH.

[0029] In Examples 1-7, different amounts (see Table 1) of L-phenylalanine and valine were added to the basic fermentation medium.

[0030] The beauveria bassiana fermentation units in Examples 1-7 are listed in Table 1 after being cultured at 28°C for 7 days.

[0031] Table 1. Beauveria bassiana shake-flask fermentation units with L-phenylalanine and valine added to the basal medium. Example 1 L-Phenylanine 1g / L + Valine 7g / L 1497 Example 2 L-Phenylanine 2g / L + Valine 5g / L 1875 Example 3 L-Phenylanine 3g / L + Valine 4g / L 2392 Example 4 L-Phenylanine 4g / L + Valine 4g / L 2448 Example 5 L-Phenylanine 4g / L + Valine 2g / L 2090 Example 6 L-Phenylanine 4g / L + Valine 3g / L 2499 Example 7 L-Phenylalanine 7g / L + Valine 0.8g / L 1623 As shown in Table 1, the simultaneous addition of L-phenylalanine and valine to the basal culture medium significantly improved the shake-flask fermentation unit of Beauveria bassiana compared to existing technologies. Within the addition range shown in Table 1, from 2 g / L L-phenylalanine + 5 g / L valine to 7 g / L L-phenylalanine + 0.8 g / L valine, the shake-flask fermentation unit of Beauveria bassiana was significantly increased. In particular, the addition range from 3 g / L L-phenylalanine + 4 g / L valine to 4 g / L L-phenylalanine + 3 g / L valine resulted in a substantial increase in the shake-flask fermentation unit of Beauveria bassiana.

[0032] II. Beauveria bassiana shake-flask fermentation levels of L-phenylalanine and valine at different addition times: Examples 8-11

[0033] The seed culture from Example 1 was inoculated into the shake-flask fermentation medium at a volume ratio of 10%, and the shaker speed was 220 RPM. During fermentation, in Examples 8-11, L-phenylalanine 4 g / L and valine 3.5 g / L were added at 24 h, 48 h, 72 h, and 96 h of culture, respectively.

[0034] The fermentation medium consisted of: 50 g / L glucose, 25 g / L maltodextrin, 20 g / L peptone, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, 3 g / L calcium carbonate, and natural pH.

[0035] Incubate at 28℃ for 7 days. The fermentation units of beauveria bassiana in Examples 8-11 are shown in Table 2.

[0036] Table 2. Beauveria bassiana shake-flask fermentation units with different supplementation times for L-phenylalanine and valine. Example 8 24h L-Phenylalanine 4g / L + Valine 3.5g / L 2621 Example 9 48h L-Phenylalanine 4g / L + Valine 3.5g / L 2548 Example 10 72h L-Phenylalanine 4g / L + Valine 3.5g / L 2401 Example 11 96h L-Phenylalanine 4g / L + Valine 3.5g / L 1458 III. Beauveria bassiana fermentation levels at different addition rates (rates) of L-phenylalanine and valine: Examples 12-17

[0037] The shake flask seed culture from Example 1 was inoculated into a 15L seed tank (the seed tank culture medium was the same as the shake flask seed culture medium) at a volume ratio of 5%, with an actual filling volume of 10L. The culture temperature was controlled at 28.0±0.5℃, the tank pressure at 0.045-0.065MPa, the aeration rate at 0.5-1.0VVM, the initial stirring speed at 200RPM, and the stirring speed at 200-550RPM during the culture process. The dissolved oxygen was controlled at above 20%, and the seed culture was obtained after 26 hours of culture.

[0038] The seed culture from the seed tank was inoculated into a 50L fermenter at a volume ratio of 10% (actual volume: 30L). The fermentation temperature was controlled at 28.0±0.5℃, the tank pressure at 0.045-0.065MPa, the aeration rate at 0.5-1.0VVM, and the initial stirring speed at 200RPM. During the cultivation process, the stirring speed was controlled at 200-550RPM to maintain dissolved oxygen above 20%; the total sugar content was controlled at 0.5-2.0%.

[0039] After fermentation for 24 hours, L-phenylalanine and valine were continuously added to the culture medium. The addition rates of L-phenylalanine and valine were different in Examples 12-17 (see Table 3), and the addition time was 96 hours.

[0040] The fermenter culture medium consisted of: 50 g / L glucose, 25 g / L maltodextrin, 20 g / L peptone, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, and 3 g / L calcium carbonate, with a natural pH.

[0041] The fermentation cycle is 7 days. The fermentation units of beauveria bassiana in Examples 12-17 are shown in Table 3.

[0042] Table 3. Beauveria bassiana fermentation units with different addition rates (rates) of L-phenylalanine and valine. Example 12 L-Phenylalanine 0.01 g / L·h + Valine 0.07 g / L·h 2166 Example 13 L-Phenylalanine 0.035 g / L·h + Valine 0.055 g / L·h 2678 Example 14 L-Phenylalanine 0.055 g / L·h + Valine 0.04 g / L·h 2925 Example 15 L-Phenylalanine 0.055 g / L·h + Valine 0.035 g / L·h 2789 Example 16 L-Phenylalanine 0.075 g / L·h + Valine 0.02 g / L·h 2449 Example 17 L-Phenylalanine 0.09 g / L·h + Valine 0.009 g / L·h 2212

Claims

1. A fermentation culture method for beauverin, comprising culturing a fertile Fusarium proliferatum strain to obtain a seed culture, then inoculating the seed culture into a basal fermentation medium for liquid fermentation culture, with a fermentation cycle of 7 days, characterized in that, Add 1-7 g / L of L-phenylalanine and 0.8-7 g / L of valine to the basal fermentation medium, or supplement L-phenylalanine and valine during fermentation. Supplementation during fermentation can be done either once or continuously. A single supplementation involves adding 1-7 g / L of L-phenylalanine and 0.8-7 g / L of valine to the fermentation broth after 24-96 hours of fermentation. Continuous supplementation involves continuously adding L-phenylalanine and valine starting after 24 hours of fermentation. The acid was maintained for 96 hours. The replenishment rate of L-phenylalanine was 0.01 g / L·h to 0.09 g / L·h, and the replenishment rate of valine was 0.009 g / L·h to 0.07 g / L·h. The composition of the basal fermentation medium was: glucose 40-70 g / L, maltodextrin 10-30 g / L, peptone 10-30 g / L, potassium dihydrogen phosphate 5-15 g / L, sodium nitrate 1-3 g / L, and calcium carbonate 1-4 g / L. The fertile Fusarium proliferatum was Fusarium proliferatum LF601, with the preservation number CGMCC 3.1777.

2. The fermentation culture method for beautifier according to claim 1, characterized in that, The basic fermentation medium consists of: 50 g / L glucose, 25 g / L maltodextrin, 20 g / L peptone, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, and 3 g / L calcium carbonate.

3. The fermentation culture method for beautifier according to any one of claims 1-2, characterized in that, The fertile Fusarium proliferatum strain was cultured in slant agar, shake flask, and seed tank to obtain seed culture. This seed culture was then inoculated into a basal fermentation medium for fermentation. The seed tank culture conditions were as follows: temperature controlled at 28.0±0.5℃, pressure at 0.045-0.065 MPa, aeration rate at 0.5-1.0 VVM, stirring speed at 200-550 RPM, dissolved oxygen above 20%, and cultured for 26 hours to obtain the seed culture. The fermentation conditions were as follows: fermentation temperature controlled at 28.0±0.5℃, pressure at 0.045-0.065 MPa, aeration rate at 0.5-1.0 VVM, stirring speed at 200-550 RPM, dissolved oxygen above 20%, and total sugar content between 0.5% and 2.0%.