Streptomyces cinnamomi and application thereof in fermentation production of monensin

Abstract

The invention belongs to the technical field of biological fermentation, and in particular relates to streptomyces cinnamomi MN-HS-01 and an application of the streptomyces cinnamomi MN-HS-01 in fermentation production of monensin. The streptomyces cinnamomi is preserved in the China Center for Type Culture Collection on July 1, 2024, and the preservation number of the streptomyces cinnamomi is CCTCC NO: M 20241414. The invention further discloses a culture medium and a culture method for producing monensin through fermentation of the strain. By means of the culture process, the bacterium concentration can be remarkably improved, meanwhile, the dissolved oxygen requirement in the fermentation process is met, and the cost is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to Streptomyces cinnamomi and application thereof in fermentation production of monensin. Background Art

[0002] Monensin, also known as monensin and rumenin, is an ionic polyether antibiotic. It is usually used to treat coccidiosis in chickens, sheep, rabbits and other poultry. Its main mechanism of action is to interfere with the Na + and K + The normal permeability of the cells eventually leads to excessive swelling, rupture and death of the cells. At the same time, it can also improve the rumen parameters of ruminants, increase food utilization and increase animal weight.

[0003] Monensin is an intracellular product. Maintaining a high bacterial concentration is beneficial to the production of antibiotics by the bacteria. However, high bacterial concentration leads to a higher demand for dissolved oxygen. During the fermentation of monensin, lack of oxygen will seriously interfere with bacterial growth and production. Therefore, it is necessary to balance the relationship between bacterial concentration and dissolved oxygen, maintaining a high bacterial concentration while ensuring oxygen supply in the fermentation tank.

[0004] Therefore, it is urgent to screen out strains that have obvious advantages in the fermentation production of monensin. Summary of the invention

[0005] In order to solve the above problems, the present invention provides a Streptomyces cinnamomi and application thereof in fermentation production of monensin.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A Streptomyces cinnamonensis MN-HS-01, wherein the Streptomyces cinnamonensis MN-HS-01 has been deposited in the China Center for Type Culture Collection on July 1, 2024, with a deposit number of CCTCC NO: M 20241414.

[0008] The present invention also provides the use of the Streptomyces cinnamonensis MN-HS-01 in fermentation production of monensin.

[0009] Furthermore, the application is to utilize the above-mentioned Streptomyces cinnamonensis MN-HS-01 to ferment and produce monensin, and the specific production process steps are as follows:

[0010] 1) Primary seed culture: sterilize the primary seed culture medium, cool it to 30-35° C., and keep the pressure with sterile air. Then, under flame protection, inoculate the seed solution of Streptomyces cinnamonensis mother bottle cultured with the Streptomyces cinnamonensis MN-HS-01 into the primary seed culture medium at an inoculation rate of 0.2%-0.4% to perform primary seed culture to obtain the primary seed solution. The seed indicators of the seed solution of Streptomyces cinnamonensis mother bottle are as follows: bacterial concentration of 5-15%, pH value higher than 7.20, no contamination by other bacteria, and culture time of 20-30 hours.

[0011] 2) Secondary seed culture: sterilize the secondary seed culture medium, cool it to 30-35°C, and keep it under pressure with sterile air. Then transfer the cultured primary seed solution into the secondary seed culture medium for secondary seed culture to obtain the secondary seed solution. The transfer ratio is controlled at 8-12%. The transfer indicators of the primary seed solution are: bacterial concentration 15-25%, pH value higher than 7.20, no contamination by other bacteria, and culture time 20-25h.

[0012] 3) Tertiary seed culture: sterilize the tertiary seed culture medium, cool it to 30-35°C, and keep it under pressure with sterile air. Then transfer the cultured secondary seed solution into the tertiary seed tank for tertiary seed culture to obtain the tertiary seed solution. The transfer ratio is controlled at 8-12%. The transfer indicators of the secondary seed solution are: bacterial concentration 25-35%, pH value higher than 7.20, no contamination by other bacteria, and culture time 20-25h.

[0013] 4) Fermentation culture: sterilize the fermentation medium, cool it to 30-35°C, and keep the pressure with sterile air, then transfer the cultured third-level seed liquid into the fermentation medium for fermentation culture to obtain fermentation liquid, and control the transfer ratio at 10-15%; the third-level seed liquid transfer index: bacterial concentration 40-50%, pH value higher than 7.20, no contamination by other bacteria, and culture time 30-35h;

[0014] Furthermore, the primary seed culture medium is composed of the following raw materials in mass percentage based on a total amount of 100%: 1.4-1.6% bean cake powder, 0.2-0.4% yeast powder, 0.3-0.5% glucose, 1-2% dextrin, 0.1-0.2% light calcium carbonate, and the balance is water;

[0015] The secondary seed culture medium is composed of the following raw materials in mass percentage based on a total amount of 100%: 1.4-1.6% bean cake powder, 0.2-0.4% yeast powder, 0.3-0.5% glucose, 1-2% dextrin, 0.1-0.2% light calcium carbonate, and the balance is water;

[0016] The three-stage seed culture medium is composed of the following raw materials in mass percentage based on a total amount of 100%: 2-4% glucose, 2-4% soybean meal, 2-3% soybean oil, 0.8-1.5% methyl oleate, 0.1-0.3% sodium nitrate, 0.1-0.3% sodium sulfate, 0.01-0.02% ferrous sulfate, 0.03-0.05% manganese chloride, 0.0001-0.0002% vitamin C, 0.05-0.10% aluminum sulfate, 0.005-0.010% dipotassium hydrogen phosphate, 0.1-0.5% corn steep liquor, 0.2-0.5% light calcium carbonate, and the balance is water;

[0017] The fermentation medium is composed of the following raw materials in mass percentage based on a total amount of 100%: 2-4% glucose, 2-4% soybean cake powder, 2-3% soybean oil, 0.8-1.5% methyl oleate, 0.1-0.3% sodium nitrate, 0.1-0.3% sodium sulfate, 0.01-0.02% ferrous sulfate, 0.03-0.05% manganese chloride, 0.0001-0.0002% vitamin C, 0.05-0.10% aluminum sulfate, 0.005-0.010% dipotassium hydrogen phosphate, 0.1-0.5% corn steep liquor, 0.2-0.5% light calcium carbonate, and the balance is water.

[0018] Furthermore, the method for obtaining the mother bottle seed liquid of Streptomyces cinnamonensis is as follows: the above-mentioned Streptomyces cinnamonensis MN-HS-01 is inoculated into the mother bottle seed liquid culture medium, the culture temperature is 30-35°C, the rotation speed is 220r / min, and the culture time is 20-30h to obtain the mother bottle seed liquid of Streptomyces cinnamonensis, wherein the mother bottle seed liquid culture medium is composed of the following raw materials in mass percentage based on the total amount of 100%: 0.5% glucose, 2% dextrin, 1.5% bean cake powder, 0.25% yeast powder, and the balance is water.

[0019] Furthermore, the first-level seed culture conditions are: tank pressure 0.03-0.05MPa; tank temperature 30-35°C; by adjusting the flow rate and stirring, the dissolved oxygen is controlled to be not less than 20%; the second-level seed culture conditions are: tank pressure 0.03-0.05MPa; tank temperature 30-35°C; by adjusting the flow rate and stirring, the dissolved oxygen is controlled to be not less than 20%; the third-level seed culture conditions are: tank pressure 0.03-0.05MPa; tank temperature 30-35°C; by adjusting the flow rate and stirring, the dissolved oxygen is controlled to be not less than 20%.

[0020] Furthermore, the fermentation culture conditions in step 4) are:

[0021] 1) Temperature: 30-35°C;

[0022] 2) Stirring speed: 0-100h: the speed is controlled at 100r / min; 101-200h: the speed is controlled at 120r / min; 201h-end of fermentation: the speed is controlled at 90r / min;

[0023] 3) Tank pressure: 0.03-0.05MPa;

[0024] 4) pH control: pH is controlled at 6.0-7.0 during fermentation;

[0025] 5) Sterility test: During the fermentation process, bacteria test is performed to ensure that there are no other bacteria;

[0026] 6) Dissolved oxygen control: 0-100h: dissolved oxygen is controlled at no less than 20%; 101-200h: dissolved oxygen is controlled at more than 25%; 201h-end of fermentation: dissolved oxygen is controlled at more than 30%;

[0027] 7) Fatty acid content: during the fermentation process, the fatty acid content is controlled at 2-3% by adding soybean oil; 24 hours before tanking, the addition of soybean oil is stopped to control the fatty acid content of tanking to be less than 1.0%.

[0028] 8) Ventilation ratio: 0.5-2.5vvm.

[0029] 9) Release standard: Fermentation is terminated when the bacterial concentration reaches 40-60%, the fatty acid content is lower than 1.0%, the fermentation titer is greater than 40000U / mL, the pH value is 6.5-7.5, and the culture time is 300-450h.

[0030] Furthermore, the step 4) also includes the step of adding phosphate to the fermentation broth, the adding time is 24-200 hours after the start of fermentation culture, and the adding method is one-time addition or flow addition; the amount of phosphate added is 0.01%-1% of the mass of the fermentation broth, and the phosphate is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.

[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0032] The method of the present invention is simple and easy to implement, and can significantly increase the bacterial concentration during the fermentation process, while meeting the dissolved oxygen demand during the fermentation process and improving the pot titer. Through the culture method of the present invention, after adding phosphate to the fermentation liquid, the pot titer reaches a maximum of 52255U / mL, which is 29.88% higher than that without feeding. At the same time, after the bacterial concentration is increased, the dry weight of the fermentation liquid is increased, and the auxiliary materials required to be added when preparing the premix are reduced. Therefore, the present invention has obvious economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1The hyphae morphology of the Streptomyces cinnamonensis MN-HS-01 strain observed under a microscope in Example 1;

[0034] Figure 2 This is the result of liquid chromatography detection of shake flask fermentation of Streptomyces cinnamonensis MN-HS-01 in Example 1. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with embodiments, and the embodiments of the present invention include but are not limited to the following embodiments.

[0036] Example 1 Screening and Identification of Streptomyces cinnamonensis MN-HS-01

[0037] Five monensin-producing Streptomyces strains screened from the soil of Xiaogan City, Hubei Province and preserved by Hubei Animal Health Products Bioengineering Technology Research Center were initially screened. Three strains with good growth were selected as starting strains.

[0038] NTG mutagenesis: The starting strain was inoculated into the plate culture medium and cultured at 33℃ for 9 days. After the spores matured, 10mL of physiological saline was added to wash the spores, and the spores were poured into a triangular flask filled with glass beads and shaken to disperse the spore suspension as much as possible. Nitrosoguanidine (NTG) was added to make the final concentration of NTG 1 mg / mL, and the treatment was 30 minutes. After centrifugation and washing with physiological saline for 3 times, the plate culture medium was diluted and coated on the plate culture medium and cultured at 33℃ for 9 days. The plate culture medium formula is based on a total amount of 100%, and is composed of the following raw materials in mass percentage: 2% starch, 2% agar powder, 0.2% potassium nitrate, 0.10% magnesium sulfate, 0.2% ferrous sulfate, 0.05% sodium chloride, 0.05% dipotassium hydrogen phosphate, and the balance is water.

[0039] Initial screening of inhibition zones: Take out the colonies cultured for 9 days together with the agar below with a cork puncher, transfer to a centrifuge tube containing 1mL 90% methanol (volume concentration, the same below), and ultrasonicate for 30min (ultrasonic frequency 40kHz, ultrasonic input power 400W, the same below) to obtain a methanol extract. Select a plate prepared with a plate culture medium, use 0.1mL of Bacillus subtilis liquid with a bacterial concentration of 5% as an indicator bacteria, and spread it evenly on the plate. Pipette 10μL of methanol extract onto the plate culture medium mixed with the indicator bacteria Bacillus subtilis, culture at 33℃ for 4d, and measure the size of the inhibition zone respectively.

[0040] Shake flask rescreening: Select single colonies with larger inhibition zones and transfer them to shake flask medium for rescreening. 2The agar block full of spores was inoculated into the shaking flask culture medium. The shaking flask culture temperature was 33°C, the rotation speed was 220r / min, and the culture time was 12d. After the culture was completed, the fermentation liquid was obtained and the fermentation titer was tested by sampling. The results are shown in Table 1. The shaking flask culture medium, based on the total amount of 100%, was composed of the following raw materials in mass percentage: 3% glucose, 4% soybean meal, 2.5% soybean oil, 1.5% methyl oleate, 0.11% sodium nitrate, 0.1% sodium sulfate, 0.02% ferrous sulfate, 0.03% manganese chloride, 0.0001% vitamin C, 0.10% aluminum sulfate, 0.005% dipotassium hydrogen phosphate, 0.5% corn steep liquor, 0.4% light calcium carbonate, and the balance was water.

[0041] The sample treatment method is as follows: take 1 mL of fermentation liquid to a 100 mL volumetric flask, add an appropriate amount of 90% methanol, and ultrasonicate for 30 minutes. After the ultrasonication, use 90% methanol to make the volume 100 mL, and filter through an organic microporous filter to obtain an extract for use.

[0042] The extract was subjected to liquid chromatography detection. The liquid chromatography detection conditions were as follows: methanol-glacial acetic acid-water (volume ratio 94:0.1:6) was used as the mobile phase, methanol-sulfuric acid-vanillin (95:2:3) (v:v:w) was used as the derivatizing agent, the flow rates of the mobile phase and the derivatizing agent were both controlled at 0.7 mL / min, the reaction temperature was 98°C, a C18 chromatographic column was used, the detection wavelength was 520 nm, and the injection volume was 20 μL. The results of the HPLC detection are shown in Figure 2 .

[0043] Table 1 Fermentation titers of 20 strains after NTG mutagenesis by shake flask screening

[0044] Strain No. Titer (U / mL) Strain No. Titer (U / mL) 1 14709 11 21089 2 21570 12 19058 3 16572 13 13863 4 16759 14 17449 5 26287 15 31188 6 18593 16 24897 7 18864 17 29608 8 29663 18 27596 9 35081 19 25939 10 16374 20 20141

[0045] After the determination, a strain with the highest fermentation titer was selected and numbered MN-HS-01. The 16SrDNA sequence of the strain was determined, and its 16S rDNA sequence is shown in the sequence table (SEQ ID NO: 1). The identification results showed that the strain belonged to Streptomyces cinnamonensis. The Streptomyces cinnamonensis MN-HS-01 was deposited in the China Center for Type Culture Collection on July 1, 2024, with a deposit number of CCTCC NO: M20241414. The hyphae morphology of the Streptomyces cinnamonensis MN-HS-01 strain observed under a microscope can be found in Figure 1 , and its 16S rDNA sequence is as follows (SEQ ID NO: 1):

[0046]

[0047] Comparative Example 1

[0048] The Streptomyces cinnamonensis MN-HS-01 in Example 1 was inoculated into a plate culture medium and cultured at 33° C. for 9 days. The plate culture medium formula was based on a total amount of 100%, and was composed of the following raw materials in percentage by mass: 2% starch, 2% agar powder, 0.2% potassium nitrate, 0.10% magnesium sulfate, 0.2% ferrous sulfate, 0.05% sodium chloride, 0.05% dipotassium hydrogen phosphate, and the balance was water.

[0049] Use the inoculation shovel to dig out 1cm of the plate culture medium 2 The well-grown Streptomyces cinnamomi is added to the mother bottle seed liquid culture medium, the culture temperature is 33°C, the rotation speed is 220r / min, and the culture time is 30h to obtain the mother bottle seed liquid of Streptomyces cinnamomi. At this time, the cell concentration of the mother bottle seed liquid is 13% and the pH value is 7.42. The mother bottle seed liquid culture medium is composed of the following raw materials in mass percentage based on the total amount of 100%: glucose 0.5%, dextrin 2%, bean cake powder 1.5%, yeast powder 0.25%, and the balance is water.

[0050] Preparation of primary seed culture medium: The volume of the seed tank is 150 L, and the effective volume is 100 L. The culture medium is composed of the following raw materials in mass percentage based on the total amount of 100%: soybean cake powder 1.4%, yeast powder 0.2%, glucose 0.4%, dextrin 1%, light calcium carbonate 0.2%, and the balance is water.

[0051] Sterilize the primary seed culture medium and cool it to 33°C, and keep it under pressure with sterile air. Sterilized culture medium: 280 μg / mL ammonia nitrogen, 45 μg / mL dissolved phosphorus, 36.5 mg / mL total sugar.

[0052] Then, under the protection of flame, the cultured cinnamon Streptomyces mother bottle seed liquid was added to the first-level seed tank for cultivation to obtain the first-level seed liquid, with an inoculation amount of 0.3%. First-level seed cultivation: tank pressure 0.03-0.05MPa; tank temperature 33℃; air flow 15m 3 / h, stirring speed 0-100r / min, by adjusting the flow and stirring, the dissolved oxygen is controlled to be not less than 20%. The first-level seed culture is completed, the bacterial concentration is 20%; the pH value is 7.35; there is no contamination from other bacteria; the culture time is 25h.

[0053] Secondary seed culture medium preparation: Seed tank volume 1.5m 3 , effective volume is 1.0m 3The culture medium is composed of the following raw materials in percentage by mass based on the total amount of 100%: 1.4% bean cake powder, 0.2% yeast powder, 0.4% glucose, 1% dextrin, 0.2% light calcium carbonate, and the balance is water.

[0054] Sterilize the secondary seed culture medium, cool it to 33°C, and keep it under pressure with sterile air. Sterilized culture medium: ammonia nitrogen 105μg / mL, dissolved phosphorus 30μg / ml, total sugar 33.1mg / mL.

[0055] Transfer all the first-level seed liquid into the second-level seed tank for cultivation to obtain the second-level seed liquid. Second-level seed cultivation conditions: tank pressure 0.03-0.05MPa; tank temperature 33℃; air flow 50-100m 3 / h, stirring speed 0-150r / min, by adjusting the flow and stirring, the dissolved oxygen is controlled to be not less than 20%. After the second-level seed culture is completed, the bacterial concentration is 29%; the pH value is 7.22; there is no contamination from other bacteria; the culture time is 20h.

[0056] Preparation of tertiary seed culture medium: Seed tank volume 25m 3 , effective volume is 12.5m 3 The culture medium is composed of the following raw materials in percentage by mass based on a total amount of 100%: 4% glucose, 3% soybean meal, 3% soybean oil, 1.5% methyl oleate, 0.1% sodium nitrate, 0.1% sodium sulfate, 0.02% ferrous sulfate, 0.03% manganese chloride, 0.0001% vitamin C, 0.05% aluminum sulfate, 0.005% dipotassium hydrogen phosphate, 0.5% corn steep liquor, 0.2% light calcium carbonate, and the balance is water.

[0057] Sterilize the third-level seed culture medium, cool it to 33°C, and keep it under pressure with sterile air. Sterilized culture medium: ammonia nitrogen 210μg / mL, dissolved phosphorus 83μg / ml, total sugar 57.2mg / mL.

[0058] Transfer all the secondary seed liquid into the third-level seed tank for cultivation to obtain the third-level seed liquid. Third-level seed cultivation conditions: tank pressure 0.03-0.05MPa; tank temperature 33℃; air flow 2000-3000m 3 / h, stirring speed 0-100r / min, by adjusting the flow and stirring, the dissolved oxygen is controlled to be not less than 20%. After the third-level seed culture, the bacterial concentration is 40%; the pH value is 7.49; there is no contamination from other bacteria; the culture time is 30h.

[0059] Fermentation medium preparation: Fermentation tank volume 165m 3 , effective volume is 120m 3The culture medium is composed of the following raw materials in percentage by mass based on a total amount of 100%: 4% glucose, 3% soybean meal, 3% soybean oil, 0.8% methyl oleate, 0.1% sodium nitrate, 0.2% sodium sulfate, 0.02% ferrous sulfate, 0.03% manganese chloride, 0.0001% vitamin C, 0.05% aluminum sulfate, 0.005% dipotassium hydrogen phosphate, 0.5% corn steep liquor, 0.2% light calcium carbonate, and the balance is water.

[0060] First, sterilize the fermentation medium, cool it to 33°C, and keep it under pressure with sterile air. Then transfer all the third-level seed liquid into the fermentation tank for fermentation to obtain the fermentation liquid. The medium after sterilization: ammonia nitrogen 209μg / mL, dissolved phosphorus 77μg / ml, total sugar 52.8mg / mL.

[0061] Fermentation culture control method:

[0062] 1) Temperature: 33°C;

[0063] 2) Stirring speed: 0-100h: the speed is controlled at 100r / min; 101-200h: the speed is controlled at 120r / min; 201h-end of fermentation: the speed is controlled at 90r / min.

[0064] 3) Tank pressure: 0.03-0.05MPa;

[0065] 4) pH control: pH is controlled at 6.0-7.0 during fermentation;

[0066] 5) Sterility test: During the fermentation process, bacteria test is performed to ensure that there are no other bacteria;

[0067] 6) Dissolved oxygen control: 0-100h, dissolved oxygen is controlled at more than 20%; 101-200h, dissolved oxygen is controlled at more than 25%; 201h-end of fermentation: dissolved oxygen is controlled at more than 30%.

[0068] 7) Fatty acid content: during the fermentation process, the fatty acid content was controlled at 2-3% by adding soybean oil. The addition of soybean oil was stopped 24 hours before tank release to control the fatty acid content below 1.0%.

[0069] At the end of the fermentation culture, the pH value was 6.8, the fatty acid was 0.8%, the bacterial concentration was 45%, the fermentation titer was 40234 U / mL, and the fermentation period was 400 h.

[0070] Embodiment 2-6

[0071] In Example 2-6, except for adding the step of adding phosphate to the fermentation broth during the fermentation culture process, the other culture processes are the same as those in Comparative Example 1, and the concentration of the added phosphate aqueous solution is 5wt%. The effects of different phosphate addition methods on the fermentation titer of each example are shown in Table 2.

[0072] Table 2 Effects of different phosphate addition methods on fermentation titer

[0073]

[0074]

[0075] As can be seen from Table 2, after phosphate supplementation, the bacterial concentration of the fermentation broth is significantly increased. Monensin is an intracellular product, and an increase in bacterial concentration will be beneficial to the increase in monensin production. However, a higher bacterial concentration also leads to a higher dissolved oxygen demand, and new monensin is basically no longer produced under hypoxia. However, through the cultivation process of the present invention, the potency of the tank is also significantly improved, and this process can meet the dissolved oxygen demand during the fermentation process.

[0076] The above embodiment is only one of the preferred implementation modes of the present invention and should not be used to limit the protection scope of the present invention. Any changes or modifications that are made to the main design concept and spirit of the present invention and have no substantive significance, and the technical problems they solve are still consistent with the present invention, should be included in the protection scope of the present invention.

Claims

1. A Streptomyces cinnamomi ( Streptomyces cinnamonensis ) MN-HS-01, characterized in that, The Streptomyces cinnamomi ( Streptomyces cinnamonensis ) MN-HS-01 was deposited in the China Center for Type Culture Collection on July 1, 2024, with the deposit number CCTCC NO: M 20241414.

2. The Streptomyces cinnamomi of claim 1 ( Streptomyces cinnamonensis ) Application of MN-HS-01 in fermentation production of monensin.

3. The use according to claim 2, characterized in that: The application is to use the Streptomyces cinnamomi described in claim 1 ( Streptomyces cinnamonensis ) MN-HS-01 fermentation produces monensin, the specific production process steps are: 1) Primary seed culture: sterilize the primary seed culture medium, cool it to 30-35°C, and keep it under pressure with sterile air. Then, under flame protection, culture the Streptomyces cinnamomi ( Streptomyces cinnamonensis ) The seed liquid of Streptomyces cinnamomi cultured in the mother bottle of MN-HS-01 is inoculated into the primary seed culture medium at an inoculation rate of 0.2%-0.4% to perform primary seed culture to obtain the primary seed liquid; the seed indicators of the seed liquid of Streptomyces cinnamomi cultured in the mother bottle are as follows: the bacterial concentration is 5-15%, the pH value is higher than 7.20, there is no contamination by other bacteria, and the culture time is 20-30h; 2) Secondary seed culture: sterilize the secondary seed culture medium, cool it to 30-35℃, and keep it under pressure with sterile air. Then transfer the cultured primary seed solution into the secondary seed culture medium for secondary seed culture to obtain the secondary seed solution. The transfer ratio is controlled at 8-12%. The transfer indicators of the primary seed solution are: bacterial concentration 15-25%, pH value higher than 7.20, no contamination by other bacteria, and culture time 20-25h. 3) Tertiary seed culture: sterilize the tertiary seed culture medium, cool it to 30-35℃, and keep it under pressure with sterile air. Then transfer the cultured secondary seed liquid into the tertiary seed tank for tertiary seed culture to obtain the tertiary seed liquid. The transfer ratio is controlled at 8-12%. The transfer indicators of the secondary seed liquid are: bacterial concentration 25-35%, pH value higher than 7.20, no contamination by other bacteria, and culture time 20-25h. 4) Fermentation culture: first sterilize the fermentation medium, cool it to 30-35℃, and keep it under pressure with sterile air, then transfer the cultured tertiary seed liquid into the fermentation medium for fermentation culture to obtain fermentation liquid, and control the transfer ratio at 10-15%; tertiary seed liquid transfer indicators: bacterial concentration 40-50%, pH value higher than 7.20, no contamination by other bacteria, and culture time 30-35h.

4. The use according to claim 3, characterized in that: The primary seed culture medium is composed of the following raw materials in percentage by mass based on a total amount of 100%: 1.4-1.6% bean cake powder, 0.2-0.4% yeast powder, 0.3-0.5% glucose, 1-2% dextrin, 0.1-0.2% light calcium carbonate, and the balance is water; The secondary seed culture medium is composed of the following raw materials in percentage by mass based on a total amount of 100%: 1.4-1.6% bean cake powder, 0.2-0.4% yeast powder, 0.3-0.5% glucose, 1-2% dextrin, 0.1-0.2% light calcium carbonate, and the balance is water; The third-level seed culture medium is composed of the following raw materials in mass percentage based on a total amount of 100%: 2-4% glucose, 2-4% soybean meal, 2-3% soybean oil, 0.8-1.5% methyl oleate, 0.1-0.3% sodium nitrate, 0.1-0.3% sodium sulfate, 0.01-0.02% ferrous sulfate, 0.03-0.05% manganese chloride, 0.0001-0.0002% vitamin C, 0.05-0.10% aluminum sulfate, 0.005-0.010% dipotassium hydrogen phosphate, 0.1-0.5% corn steep liquor, 0.2-0.5% light calcium carbonate, and the balance is water; The fermentation medium is composed of the following raw materials in mass percentage based on a total amount of 100%: 2-4% glucose, 2-4% soybean meal, 2-3% soybean oil, 0.8-1.5% methyl oleate, 0.1-0.3% sodium nitrate, 0.1-0.3% sodium sulfate, 0.01-0.02% ferrous sulfate, 0.03-0.05% manganese chloride, 0.0001-0.0002% vitamin C, 0.05-0.10% aluminum sulfate, 0.005-0.010% dipotassium hydrogen phosphate, 0.1-0.5% corn steep liquor, 0.2-0.5% light calcium carbonate, and the balance is water.

5. The use according to claim 3, characterized in that: The method for obtaining the seed liquid of the mother bottle of Streptomyces cinnamomi is as follows: Streptomyces cinnamonensis ) MN-HS-01 is inoculated into the mother bottle seed liquid culture medium, the culture temperature is 30-35℃, the rotation speed is 220r / min, and the culture time is 20-30h to obtain the cinnamon Streptomyces mother bottle seed liquid. The mass percentage of the mother bottle seed liquid culture medium is based on the total amount 100%, and it is composed of the following raw materials in mass percentage: glucose 0.5%, dextrin 2%, bean cake powder 1.5%, yeast powder 0.25%, and the balance is water.

6. The use according to claim 3, characterized in that: The first-level seed culture conditions are: tank pressure 0.03-0.05MPa; tank temperature 30-35°C; by adjusting the flow rate and stirring, the dissolved oxygen is controlled to be not less than 20%; The secondary seed culture conditions are: tank pressure 0.03-0.05MPa; tank temperature 30-35°C; by adjusting the flow rate and stirring, the dissolved oxygen is controlled to be not less than 20%; The three-stage seed culture conditions are: tank pressure 0.03-0.05MPa; tank temperature 30-35°C; by adjusting the flow rate and stirring, the dissolved oxygen is controlled to be not less than 20%.

7. The use according to claim 3, characterized in that: The fermentation culture conditions in step 4) are: 1) Temperature: 30-35℃; 2) Stirring speed: 0-100h: the speed is controlled at 100r / min; 101-200h: the speed is controlled at 120r / min; 201h-end of fermentation: the speed is controlled at 90r / min; 3) Tank pressure: 0.03-0.05MPa; 4) pH control: pH is controlled at 6.0-7.0 during fermentation; 5) Sterility test: During the fermentation process, bacteria test is performed to ensure that there are no other bacteria; 6) Dissolved oxygen control: 0-100h: dissolved oxygen is controlled at no less than 20%; 101-200h: dissolved oxygen is controlled at more than 25%; 201h-end of fermentation: dissolved oxygen is controlled at more than 30%; 7) Fatty acid content: During the fermentation process, the fatty acid content is controlled at 2-3% by adding soybean oil; 24 hours before tanking, stop adding soybean oil to control the fatty acid content below 1.0%; 8) Ventilation ratio: 0.5-2.5vvm; 9) Tank release standard: Fermentation is terminated when the bacterial concentration reaches 40-60%, the fatty acid content is lower than 1.0%, the fermentation titer is greater than 40,000 U / mL, the pH value is 6.5-7.5, and the culture time is 300-450 hours.

8. The use according to claim 3, characterized in that The step 4) also includes the step of adding phosphate to the fermentation broth, the adding time is 24-200 hours after the start of fermentation culture, and the adding method is one-time addition or flow addition; The amount of the phosphate added is 0.01%-1% of the mass of the fermentation liquid, and the phosphate is one or more of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.

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