A fermentation culture method of a 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 or valine is added to the basal fermentation medium or fermentation broth. This method can significantly increase the yield of beauveria bassiana, achieving a fermentation level of over 2100 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] 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.

[0003] 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.

[0004] 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.

[0005] 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

[0006] 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.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A fermentation culture method for beautifier: Fertile Fusarium fusarium proliferatum The 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, 1-10 g / L of L-phenylalanine or 0.8-8 g / L of valine is added to the basal fermentation medium or fermentation broth. The basal fermentation medium is a portion of the medium that does not contain L-phenylalanine or 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] It is preferable to add 3-6 g / L of L-phenylalanine or 2-6 g / L of valine.

[0011] L-phenylalanine or valine can be added directly to the basic fermentation medium, as in the shake-flask fermentation medium of Examples 1-11 where L-phenylalanine or valine was added directly. Alternatively, L-phenylalanine or valine can be added to the fermentation broth after 24-120 hours of fermentation.

[0012] The method for supplementing L-phenylalanine or valine after 24-120 hours of fermentation can be a one-time addition, as in Examples 12-19, where L-phenylalanine or valine is added to the shake-flask fermentation medium after a certain period of cultivation. Preferably, L-phenylalanine or valine is added to the shake-flask fermentation medium once within 24-72 hours of cultivation. Alternatively, L-phenylalanine or valine can be continuously added starting after 24 hours of fermentation, as in Examples 20-30, where L-phenylalanine or valine is continuously added to the fermenter medium after 24 hours of fermentation. The duration of supplementation can be 24-96 hours; preferably, the duration of supplementation is 96 hours, at which time the supplementation rate of L-phenylalanine is 0.01 g / L·h-0.1 g / L·h, or the supplementation rate of valine is 0.009 g / L·h-0.085 g / L·h; preferably, the supplementation rate of L-phenylalanine is 0.035 g / L·h-0.07 g / L·h, or preferably, the supplementation rate of valine is 0.03 g / L·h-0.065 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 described in this invention fusarium proliferatum It could be Fusarium fertilum LF601, with accession number CGMCC 3.1777.

[0016] The beneficial effects of this invention are mainly reflected in:

[0017] 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 with L-phenylalanine or valine during fermentation, and coordinating fermentation process control. This results in a substantial increase in the yield of beauveria bassiana. The invention features a simple operating process, few impurities, and stable process control, making it suitable for industrial production and conducive to its widespread application. Implementation

[0018] 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.

[0019] In this invention, there are no special restrictions on the materials used; any material source well known in the art may be used.

[0020] In this invention, the beauveria bassiana producing bacterium is a fertile Fusarium (Fusarium wiltii). fusarium proliferatum LF601.

[0021] 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.

[0022] In this invention, beauveria bassiana can be detected by high-performance liquid chromatography (HPLC) under the following conditions:

[0023] Chromatographic column: Agilent C18 column (4.6 mm × 250 mm, 5 μm);

[0024] Mobile phase: Acetonitrile:water = 8:2;

[0025] Flow rate: 1.0 mL / min;

[0026] Column temperature: 31℃;

[0027] Detection wavelength: 254nm;

[0028] Injection volume: 10 μl.

[0029] I. Beauveria bassiana shake-flask fermentation level with L-phenylalanine or valine added to the basal medium

[0030] Example 1

[0031] (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.

[0032] The slant culture medium is potato dextrose agar medium.

[0033] (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.

[0034] The shake flask seed culture medium consisted of: 20 g / L glucose, 10 g / L yeast extract, 20 g / L peptone, and natural pH.

[0035] (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.

[0036] The fermentation medium consisted of: 70 g / L glucose, 30 g / L maltodextrin, 30 g / L peptone, 1 g / L L-phenylalanine, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, and 3 g / L calcium carbonate, with a natural pH.

[0037] After incubation at 28℃ for 7 days, the fermentation unit of beauveria bassiana was 1345µg / ml.

[0038] Example 2

[0039] The shake flask 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.

[0040] The fermentation medium consisted of: 40 g / L glucose, 30 g / L maltodextrin, 10 g / L peptone, 10 g / L L-phenylalanine, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, 3 g / L calcium carbonate, and natural pH.

[0041] After incubation at 28℃ for 7 days, the fermentation unit of beauveria bassiana was 1098µg / ml.

[0042] Example 3

[0043] The shake flask 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.

[0044] The fermentation medium consisted of: 40 g / L glucose, 25 g / L maltodextrin, 10 g / L peptone, 4 g / L L-phenylalanine, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, 3 g / L calcium carbonate, and natural pH.

[0045] After incubation at 28℃ for 7 days, the fermentation unit of beauveria bassiana was 1422 µg / ml.

[0046] Example 4

[0047] The shake flask 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.

[0048] The fermentation medium consisted of: 50 g / L glucose, 25 g / L maltodextrin, 20 g / L peptone, 4 g / L L-phenylalanine, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, and 3 g / L calcium carbonate, with a natural pH.

[0049] After incubation at 28℃ for 7 days, the fermentation unit of beauveria bassiana was 1732 µg / ml.

[0050] Example 5

[0051] The shake flask 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.

[0052] The fermentation medium consisted of: 50 g / L glucose, 25 g / L maltodextrin, 20 g / L peptone, 6 g / L L-phenylalanine, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, 3 g / L calcium carbonate, and natural pH.

[0053] After incubation at 28℃ for 7 days, the fermentation unit of beauveria bassiana was 1556 µg / ml.

[0054] Example 6

[0055] The shake flask 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.

[0056] The fermentation medium consisted of: 50 g / L glucose, 20 g / L maltodextrin, 25 g / L peptone, 3 g / L L-phenylalanine, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, and 3 g / L calcium carbonate, with a natural pH.

[0057] After incubation at 28℃ for 7 days, the fermentation unit of beauveria bassiana was 1670µg / ml.

[0058] Examples 7-11

[0059] Unlike in Examples 1-6 where L-phenylalanine was added to the basal fermentation medium, valine was added to the basal medium at concentrations of 0.8, 8, 2, 6, and 3.5 g / L, respectively, with the other conditions being the same as in Example 4.

[0060] The shake-flask fermentation units of beauveria bassiana with L-phenylalanine or valine added to the basal culture medium in Examples 1-11 are shown in Table 1.

[0061] Table 1. Beauveria bassiana shake-flask fermentation units with L-phenylalanine or valine added to the basal medium.

[0062]

[0063] II. Beauveria bassiana shake-flask fermentation levels with different addition times of L-phenylalanine or valine.

[0064] Example 12

[0065] 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, L-phenylalanine was added all at once after 24 hours of culture. 4 g / L.

[0066] 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.

[0067] Incubate at 28℃ for 7 days. The fermentation unit of beauveria bassiana is 1873 µg / ml.

[0068] Example 13

[0069] 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, 4 g / L of L-phenylalanine was added once after 48 hours of culture.

[0070] 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.

[0071] Incubate at 28℃ for 7 days. The fermentation unit of beauveria bassiana is 1815 µg / ml.

[0072] Example 14

[0073] 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, 4 g / L of L-phenylalanine was added once after 72 hours of culture.

[0074] 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.

[0075] Incubate at 28℃ for 7 days. The fermentation unit of beauveria bassiana is 1797 µg / ml.

[0076] Example 15

[0077] 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, 4 g / L of L-phenylalanine was added once after 96 h of culture.

[0078] 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.

[0079] Incubate at 28℃ for 7 days. The fermentation unit of beauveria bassiana is 1358 µg / ml.

[0080] Examples 16-19

[0081] Unlike Examples 12-15, where L-phenylalanine was added to the fermentation broth once at 24, 48, 72, and 96 hours of cultivation, valine 3.5 g / L was added to the fermentation broth once at 24, 48, 72, and 96 hours of cultivation.

[0082] Table 2 shows the beauveria bassiana shake-flask fermentation units with different addition times for L-phenylalanine or valine in Examples 12-19.

[0083] Table 2. Beauveria bassiana shake-flask fermentation units with different L-phenylalanine or valine supplementation times

[0084] Example 12 24h L-Phenylanine 4g / L 1873 Example 13 48h L-Phenylanine 4g / L 1815 Example 14 72h L-Phenylanine 4g / L 1797 Example 15 96h L-Phenylanine 4g / L 1358 Example 16 24h Valine 3.5g / L 1752 Example 17 48h Valine 3.5g / L 1686 Example 18 72h Valine 3.5g / L 1521 Example 19 96h Valine 3.5g / L 1318

[0085] III. Beauveria bassiana fermentation levels at different addition rates (rates) of L-phenylalanine or valine.

[0086] Example 20

[0087] 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.

[0088] 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%.

[0089] After fermentation for 24 hours, L-phenylalanine was continuously added to the culture medium at a rate of 0.01 g / L·h for 96 hours.

[0090] 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.

[0091] The fermentation cycle was 7 days, and the fermentation unit of beauveria bassiana was 1650µg / ml.

[0092] Example 21

[0093] After fermentation for 24 hours, L-phenylalanine was continuously added to the culture medium at a rate of 0.035 g / L·h for 96 hours. Other conditions were the same as in Example 20.

[0094] 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.

[0095] The fermentation cycle was 7 days, and the fermentation unit of beauveria bassiana was 2047 µg / ml.

[0096] Example 22

[0097] After fermentation for 24 hours, L-phenylalanine was continuously added to the culture medium at a rate of 0.05 g / L·h for 96 hours. Other conditions were the same as in Example 20.

[0098] 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.

[0099] The fermentation cycle was 7 days, and the fermentation unit of beauveria bassiana was 2162 µg / ml.

[0100] Example 23

[0101] After fermentation for 24 hours, L-phenylalanine was continuously added to the culture medium at a rate of 0.07 g / L·h for 96 hours. Other conditions were the same as in Example 20.

[0102] 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.

[0103] The fermentation cycle was 7 days, and the fermentation unit of beauveria bassiana was 1877 µg / ml.

[0104] Example 24

[0105] After fermentation for 24 hours, L-phenylalanine was continuously added to the culture medium at a rate of 0.075 g / L·h for 96 hours. Other conditions were the same as in Example 20.

[0106] 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.

[0107] The fermentation cycle was 7 days, and the fermentation unit of beauveria bassiana was 1595µg / ml.

[0108] Example 25

[0109] After fermentation for 24 hours, L-phenylalanine was continuously added to the culture medium at a rate of 0.1 g / L·h for 96 hours. Other conditions were the same as in Example 20.

[0110] 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.

[0111] The fermentation cycle was 7 days, and the fermentation unit of beauveria bassiana was 1258µg / ml.

[0112] Examples 26-30

[0113] Unlike the addition of L-phenylalanine in Examples 20-25, valine was added at different rates.

[0114] Table 3 shows the different amounts of Beauveria bassiana added to the fermentation tanks when L-phenylalanine or valine were added in Examples 20-30.

[0115] Table 3. Beauveria bassiana extract supplementation at different amounts in fermentation units with added L-phenylalanine or valine.

[0116] Example 20 L-Phenylalanine 0.01 g / L·h 1650 Example 21 L-Phenylalanine 0.035 g / L·h 2047 Example 22 L-Phenylalanine 0.05 g / L·h 2162 Example 23 L-Phenylalanine 0.07 g / L·h 1877 Example 24 L-Phenylalanine 0.075 / L·h 1595 Example 25 L-Phenylalanine 0.1 g / L·h 1258 Example 26 Valine 0.009 g / L·h 1524 Example 27 Valine 0.03 g / L·h 1857 Example 28 Valine 0.04 g / L·h 1945 Example 29 Valine 0.065 g / L·h 1785 Example 30 Valine 0.085 g / L·h 1217

[0117] Comparative Example 1

[0118] The shake flask 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.

[0119] The fermentation medium consisted of: 50 g / L sucrose, 20 g / L maltodextrin, 30 g / L peptone, 5 g / L yeast extract, 10 g / L potassium dihydrogen phosphate, 2 g / L sodium nitrate, and 3 g / L calcium carbonate, with a natural pH.

[0120] After incubation at 28℃ for 7 days, the fermentation unit of beauveria bassiana was 437µg / ml.

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 1-10 g / L of L-phenylalanine to the basal fermentation medium or supplement L-phenylalanine during fermentation. Supplementation during fermentation can be done in a single addition or continuously. A single addition involves adding 1-10 g / L of L-phenylalanine to the fermentation broth at 24-96 hours of fermentation. Continuous supplementation involves adding L-phenylalanine continuously starting after 24 hours of fermentation and continuing for 96 hours at a rate of 0.01-0.1 g / L·h. The basal fermentation medium consists of: 40-70 g / L glucose, 20-30 g / L maltodextrin, 10-30 g / L peptone, 5-15 g / L potassium dihydrogen phosphate, 1-3 g / L sodium nitrate, and 1-4 g / L calcium carbonate. The fertile Fusarium proliferatum is fertile Fusarium LF601, with 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 beauverin 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%.

Images