Method for producing nemadectin by fermentation method

Abstract

The invention relates to the technical field of biological fermentation engineering, and provides a method for producing nemadectin by a fermentation method. The invention relates to a method for producing nemadectin by a fermentation method, which comprises the following steps: inoculating a nemadectin production strain seed solution into a fermentation culture medium for fermentation culture to obtain a fermentation solution containing nemadectin; the fermentation culture medium is prepared from the following components in parts by weight: 4 to 6 parts of glucose, 2 to 2.5 parts of starch, 2.5 to 3.5 parts of soybean cake powder, 0.5 to 0.8 part of cottonseed cake powder, 1 to 2 parts of fish meal, 0.72 to 1.25 parts of inorganic salt, 0.15 to 0.25 part of defoaming agent and 100 parts of water. According to the technical scheme, the problem that the fermentation content of nemadectin is low when a fermentation method is adopted to produce nemadectin in the related technology is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of biological fermentation engineering, and in particular to a method for producing nimoctin by fermentation. Background Art

[0002] Nimoctin is a 16-membered lactone compound belonging to the milbemycin family of antibiotics. It is the starting compound for the synthesis of moxidectin. Based on nimoctin, an N-OCH3 group is introduced at the C-23 position to form nimoctin, which is more lipophilic. As a safe and effective insecticide, moxidectin has many advantages over existing biopesticides. Its mechanism of action is similar to that of ivermectin, but it has higher activity than ivermectin. It has the characteristics of low dosage, high safety, non-toxicity, and non-pollution, and is not prone to drug resistance. It is a biopesticide widely favored by the industry. As a precursor of moxidectin, the production capacity of nimoctin determines the production capacity of moxidectin.

[0003] In order to further improve the competitiveness of technology and reduce the production cost of moxidectin, researchers are constantly trying new methods to increase the production capacity of nimoctin or reduce production costs. For example, patent CN104450823A discloses a method of adding sodium acetate during the fermentation process to increase the fermentation content of nimoctin. However, the addition of sodium acetate easily causes the fermentation cost to increase, and it needs to be added separately, and the feeding tank is configured separately, which increases the complexity of the process operation. Patent CN108774637A discloses a method of using Streptomyces cyanobacterium and adding L-valine during the fermentation process to produce nimoctin, but L-valine itself has a certain harmful effect on bacterial growth, especially when the local concentration is too high, it is easy to cause bacterial metabolic disorders, which is not conducive to the control of the overall fermentation metabolism, and it is possible for the bacterial metabolism to produce other impurities. Patent CN110387390A discloses a method of adding soybean oil to the fermentation medium to produce nimoctin, and the addition of soybean oil substances easily causes the fermentation liquid to become viscous, which will cause difficulties in the extraction link in the subsequent production process.

[0004] Therefore, developing a method for producing nimoctin by fermentation can further improve the production capacity of nimoctin and reduce production costs, which is of great significance for meeting people's needs. Summary of the Invention

[0005] The present invention provides a method for producing nimoctin by fermentation, which solves the problem of low nimoctin fermentation content when nimoctin is produced by fermentation in the related art.

[0006] The technical solutions of the present invention are as follows: The present invention provides a method for producing nimoctin by fermentation, comprising the following steps: inoculating a nimoctin-producing strain seed solution into a fermentation medium for fermentation culture to obtain a fermentation solution containing nimoctin; The fermentation medium comprises the following components in parts by weight: 4-6 parts of glucose, 2-2.5 parts of starch, 2.5-3.5 parts of soybean cake powder, 0.5-0.8 parts of cottonseed cake powder, 1-2 parts of fish meal, 0.72-1.25 parts of inorganic salt, 0.15-0.25 parts of defoaming agent, and 100 parts of water.

[0007] As a further technical solution, the fish meal comprises fish meal I and fish meal II in a weight ratio of 3 to 9:1; The fish meal I and fish meal II have different particle sizes.

[0008] As a further technical solution, the particle size of the fish meal I is 40-60 mesh, and the particle size of the fish meal II is 80-100 mesh.

[0009] In the fermentation method for producing nimoctin according to the present invention, the fish meal comprises fish meal I with a particle size of 40-60 mesh and fish meal II with a particle size of 80-100 mesh. The combined use of these two fish meals with different particle sizes can further increase the fermentation yield of nimoctin. This is presumably due to the fact that the strain rapidly consumes the nitrogen source provided by the smaller-particle fish meal during the initial stages of the fermentation, promoting the strain's primary metabolism and enabling faster entry into secondary metabolism, thus providing the energy required for nimoctin synthesis. Furthermore, during the nimoctin synthesis stage, the larger-particle fish meal prevents the strain from rapidly consuming the nitrogen source during the initial stages of the fermentation, thereby providing the building blocks for subsequent nimoctin synthesis. This overall increase in nimoctin fermentation yield is further enhanced. By exploring the particle size and content ratio of the two fish meals, it was found that when the weight ratio of fish meal with a particle size of 40-60 mesh to fish meal with a particle size of 80-100 mesh is 3-9:1, the fermentation yield of nimoctin can be better increased, and the fermentation content of nimoctin can reach 3167-3172 μg / mL. When the weight ratio is outside the range of 3-9:1, the effect is slightly worse.

[0010] As a further technical solution, the inorganic salt includes one or more of magnesium sulfate, zinc sulfate, and calcium carbonate.

[0011] As a further technical solution, when the inorganic salt includes magnesium sulfate, zinc sulfate and calcium carbonate, the amount of magnesium sulfate is 0.5-0.8 parts, the amount of zinc sulfate is 0.02-0.05 parts, and the amount of calcium carbonate is 0.2-0.4 parts.

[0012] As a further technical solution, the defoaming agent includes one or both of an organosilicon defoaming agent and a polyether defoaming agent.

[0013] In the method for producing nimoctin by fermentation of the present invention, the defoaming agent includes one or both of an organosilicon defoaming agent and a polyether defoaming agent, wherein the organosilicon defoaming agent can be, for example, a dimethyl silicone oil defoaming agent, and the polyether defoaming agent can be, for example, polyoxypropylene oxyethylene glycerol ether and polyoxyethylene polyoxypropylene pentaerythritol ether, preferably polyoxypropylene oxyethylene glycerol ether.

[0014] As a further technical solution, the fermentation medium also includes sodium propionate, and the weight ratio of the fish meal to the sodium propionate is 1-2:1.

[0015] In the method for producing nimoctin by fermentation of the present invention, when sodium propionate is also included in the fermentation medium, sodium propionate and fish meal are used together, and the weight ratio of fish meal to sodium propionate is adjusted to 1-2:1. This can provide a stable fermentation environment for the strain and also provide sufficient precursor substances for the synthesis of nimoctin, thereby further increasing the fermentation content of nimoctin.

[0016] As a further technical solution, the nimoctin-producing strain seed liquid is a chromogenic Streptomyces seed liquid; The Streptomyces chromogenes seed liquid accounts for 5% to 10% of the volume of the fermentation medium, for example, 5%, 6%, 7%, 8%, 9%, 10%, preferably 5%, 10%, and more preferably 10%.

[0017] As a further technical solution, during the fermentation culture, the fermentation temperature is 26~28°C, for example, it can be 26°C, 27°C, 28°C, preferably 28°C, the tank pressure is 0.03~0.04MPa, for example, it can be 0.03MPa, 0.035MPa, 0.04MPa, preferably 0.04MPa, the relative dissolved oxygen is 30%~50%, and the fermentation period is 235~240h, for example, it can be 235h, 236h, 237h, 238h, 239h, 240h, preferably 235h, 240h, more preferably 240h.

[0018] As a further technical solution, during the fermentation culture, the glucose content is controlled to be 10-20 g / L.

[0019] The working principle and beneficial effects of the present invention are: In the method for producing nimoctin by fermentation of the present invention, nimoctin is obtained by fermenting the nimoctin-producing strain seed liquid in a fermentation medium. The fermentation medium includes glucose, starch, soybean cake powder, cottonseed cake powder, fish meal, inorganic salts, defoaming agents and water, wherein glucose is used as a quick-acting carbon source to meet the energy demand of the strain growth in the early stage of fermentation and promote the rapid proliferation of mycelium. Starch is used as a slow-release carbon source and is gradually decomposed into glucose in the middle and late stages of fermentation to continuously provide a carbon source. Soybean cake powder, cottonseed cake powder and fish meal are added to the fermentation medium as nitrogen sources, which can provide an essential nitrogen source for the growth metabolism of the strain, and by reasonably limiting the component content of the nitrogen source, the strain growth is in a moderate state. Among them, the branched-chain fatty acids and various amino acids in the fish meal can provide the necessary amino acids for the metabolism of the bacterial cell, and also provide the necessary precursor substances for nimoctin, thereby promoting the synthesis of nimoctin. By adding fish meal and rationally adjusting the content of fish meal, the fermentation content of nimoctin can be increased to 3077~3110μg / mL, thereby achieving the purpose of increasing the fermentation content of nimoctin. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0021] In the following examples and comparative examples, the polyoxypropylene ethylene glycerol ether model is F-38; The Streptomyces chromogenes is DC18-01, with a deposit number of CGMCC No. 6217 and a deposit date of June 14, 2012. The published patent application document is application number 201410327571.6, with an application date of July 10, 2014. It has been deposited with a depository institution recognized by the State Intellectual Property Office for patent procedures and can be deemed to be available to the public without the need for deposit. The seed solution of the Streptomyces chromogenes was cultured according to the culture method of Example 1 in publication No. CN 104109648 A - A Streptomyces chromogenes strain with high nimoctin production and screening method thereof.

[0022] Example 1 The fermentation medium includes the following components: Glucose 4g, starch 2.0g, soybean meal 2.5g, cottonseed meal 0.5g, 50-mesh fish meal 1g, magnesium sulfate 0.5g, zinc sulfate 0.02g, calcium carbonate 0.2g, polyoxypropylene glycol ether 0.15g, water 100mL; A method for producing nimoctin by fermentation comprises the following steps: inoculating a chromogenic Streptomyces chromogenicus seed solution into a fermentation medium at a volume ratio of 5%, fermenting and culturing at 26°C and a tank pressure of 0.03 MPa for 235 hours, maintaining a relative dissolved oxygen content of 30% to 50%, to obtain a fermentation liquid containing nimoctin; wherein, during the fermentation process, the glucose content is controlled to be 10 to 20 g / L.

[0023] Example 2 The fermentation medium includes the following components: Glucose 6g, starch 2.5g, soybean meal 3.5g, cottonseed meal 0.8g, 50-mesh fish meal 2g, magnesium sulfate 0.8g, zinc sulfate 0.05g, calcium carbonate 0.4g, polyoxypropylene glycol ether 0.25g, water 100mL; A method for producing nimoctin by fermentation comprises the following steps: inoculating a chromogenic Streptomyces chromogenicus seed solution into a fermentation medium at a volume ratio of 10%, fermenting and culturing at 28°C and a tank pressure of 0.04 MPa for 240 hours, maintaining a relative dissolved oxygen content of 30% to 50%, to obtain a fermentation liquid containing nimoctin; wherein, during the fermentation process, the glucose content is controlled to be 10 to 20 g / L.

[0024] Example 3 The only difference between this embodiment and embodiment 2 is that in the fermentation medium of this embodiment, the fish meal with a particle size of 50 mesh is replaced with an equal amount of fish meal with a particle size of 90 mesh.

[0025] Example 4 The only difference between this embodiment and embodiment 2 is that in the fermentation medium of this embodiment, the amount of fish meal with a particle size of 50 mesh added is 1 g, and the amount of fish meal with a particle size of 90 mesh added is 1 g.

[0026] Example 5 The only difference between this embodiment and embodiment 2 is that in the fermentation medium of this embodiment, the amount of fish meal with a particle size of 50 mesh added is 1.9 g, and the amount of fish meal with a particle size of 90 mesh added is 0.1 g.

[0027] Example 6 The only difference between this embodiment and embodiment 2 is that in the fermentation medium of this embodiment, the amount of fish meal with a particle size of 50 mesh added is 1.5 g, and the amount of fish meal with a particle size of 90 mesh added is 0.5 g.

[0028] Example 7 The only difference between this embodiment and embodiment 2 is that in the fermentation medium of this embodiment, the amount of fish meal with a particle size of 50 mesh added is 1.8 g, and the amount of fish meal with a particle size of 90 mesh added is 0.2 g.

[0029] Example 8 The only difference between this embodiment and embodiment 7 is that in the fermentation medium of this embodiment, the fish meal with a particle size of 50 mesh is replaced by an equal amount of fish meal with a particle size of 40 mesh, and the fish meal with a particle size of 90 mesh is replaced by an equal amount of fish meal with a particle size of 80 mesh.

[0030] Example 9 The only difference between this embodiment and embodiment 7 is that in the fermentation medium of this embodiment, the fish meal with a particle size of 50 mesh is replaced by an equal amount of fish meal with a particle size of 60 mesh, and the fish meal with a particle size of 90 mesh is replaced by an equal amount of fish meal with a particle size of 100 mesh.

[0031] Example 10 The only difference between this embodiment and embodiment 4 is that in the fermentation medium of this embodiment, the fish meal with a particle size of 50 mesh is replaced by an equal amount of fish meal with a particle size of 30 mesh, and the fish meal with a particle size of 90 mesh is replaced by an equal amount of fish meal with a particle size of 70 mesh.

[0032] Example 11 The only difference between this embodiment and embodiment 4 is that in the fermentation medium of this embodiment, the fish meal with a particle size of 50 mesh is replaced by an equal amount of fish meal with a particle size of 70 mesh, and the fish meal with a particle size of 90 mesh is replaced by an equal amount of fish meal with a particle size of 120 mesh.

[0033] Example 12 The only difference between this embodiment and embodiment 7 is that the fermentation medium of this embodiment further includes 1 g of sodium propionate.

[0034] Example 13 The only difference between this embodiment and embodiment 7 is that the fermentation medium of this embodiment further includes 2 g of sodium propionate.

[0035] Comparative Example 1 The only difference between Comparative Example 1 and Example 2 is that the amount of fish meal with a particle size of 50 mesh added to the fermentation medium of this comparative example is 0.5 g.

[0036] Comparative Example 2 The only difference between Comparative Example 1 and Example 2 is that the amount of fish meal with a particle size of 50 mesh added to the fermentation medium of this comparative example is 2.5 g.

[0037] Comparative Example 3 The only difference between Comparative Example 1 and Example 2 is that no fish meal was added to the fermentation medium of this comparative example.

[0038] Comparative Example 4 The only difference between Comparative Example 1 and Example 2 is that in the fermentation medium of this comparative example, the fish meal with a particle size of 50 mesh is replaced by an equal amount of sodium propionate.

[0039] The nimoctin content in the fermentation broths obtained in Examples 1 to 13 and Comparative Examples 1 to 4 was measured. The measurement results are shown in Table 1, wherein the difference ratio with Comparative Example 3 = (nimoctin content in the example or comparative example - nimoctin content in Comparative Example 3) / nimoctin content in Comparative Example 3 × 100%.

[0040] Table 1 Nimoctin content test results of Examples 1 to 13 and Comparative Examples 1 to 4

[0041] As can be seen from Table 1, compared with Comparative Example 3, the nimoctin content in the fermentation broth obtained in Examples 1 to 13 increased significantly, indicating that the addition of fish meal to the fermentation medium can effectively increase the fermentation yield of nimoctin. Compared with Comparative Examples 1 to 2, the nimoctin content in the fermentation broth obtained in Examples 1 to 2 increased, indicating that a fish meal concentration of 1.0 to 2.0 g / mL in the fermentation medium has a better effect on increasing the fermentation yield of nimoctin. Compared with Examples 2 to 3 and Examples 10 to 11, the nimoctin content in the fermentation broth obtained in Examples 4 to 9 increased, indicating that when the particle size of the fish meal is 40 to 60 mesh and 80 to 100 mesh, the fermentation yield of nimoctin can be further increased by using two fish meals with different particle sizes. In addition, compared with Example 8, the nimoctin content in the fermentation broth obtained in Examples 12 to 13 increased, indicating that when the fermentation medium also includes sodium propionate, the fermentation yield of nimoctin can be further increased.

[0042] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for producing nimoctin by fermentation, characterized in that: The following steps are involved: inoculating the seed liquid of the nimoctin-producing strain into a fermentation medium for fermentation culture to obtain a fermentation liquid containing nimoctin; The fermentation medium comprises the following components in parts by weight: 4-6 parts of glucose, 2-2.5 parts of starch, 2.5-3.5 parts of soybean cake powder, 0.5-0.8 parts of cottonseed cake powder, 1-2 parts of fish meal, 0.72-1.25 parts of inorganic salt, 0.15-0.25 parts of defoaming agent, and 100 parts of water.

2. The method for producing nimoctin by fermentation according to claim 1, characterized in that: The fish meal comprises fish meal I and fish meal II in a weight ratio of 3 to 9:1; The fish meal I and fish meal II have different particle sizes.

3. The method for producing nimoctin by fermentation according to claim 2, characterized in that: The particle size of the fish meal I is 40-60 meshes, and the particle size of the fish meal II is 80-100 meshes.

4. The method for producing nimoctin by fermentation according to claim 1, characterized in that: The inorganic salt includes one or more of magnesium sulfate, zinc sulfate, and calcium carbonate.

5. The method for producing nimoctin by fermentation according to claim 4, characterized in that: When the inorganic salt includes magnesium sulfate, zinc sulfate and calcium carbonate, the amount of magnesium sulfate is 0.5-0.8 parts, the amount of zinc sulfate is 0.02-0.05 parts, and the amount of calcium carbonate is 0.2-0.4 parts.

6. The method for producing nimoctin by fermentation according to claim 1, characterized in that: The defoaming agent includes one or both of an organosilicon defoaming agent and a polyether defoaming agent.

7. The method for producing nimoctin by fermentation according to claim 1, characterized in that: The fermentation medium also includes sodium propionate, and the weight ratio of the fish meal to the sodium propionate is 1-2:

1.

8. The method for producing nimoctin by fermentation according to claim 1, characterized in that: The nimoctin production strain seed liquid is a chromogenic Streptomyces seed liquid; The Streptomyces chromogenes seed liquid accounts for 5% to 10% of the volume of the fermentation medium.

9. The method for producing nimoctin by fermentation according to claim 1, characterized in that: During the fermentation culture, the fermentation temperature is 26-28° C., the tank pressure is 0.03-0.04 MPa, the relative dissolved oxygen is 30%-50%, and the fermentation period is 235-240 hours.

10. The method for producing nimoctin by fermentation according to claim 1, characterized in that: During the fermentation culture, the glucose content is controlled to be 10-20 g / L.

Images