Fermentation medium and method for preparing josamycin

Abstract

The fermentation culture medium comprises a carbon source and a nitrogen source, and the carbon source is selected from one or more of soybean oil, peanut oil, rapeseed oil and olive oil; the nitrogen source is selected from one or more of corn protein powder, sesame cake powder, soybean cake powder, cottonseed cake powder, biological nitrogen and L-leucine. The fermentation medium provided by the invention can be used for efficiently and stably producing josamycin, and is simple to operate, low in cost and suitable for industrial production.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation, and particularly to a fermentation medium for preparing josamycin and a method thereof. Background Art

[0002] Josamycin is a broad-spectrum antibiotic, and its antibacterial spectrum is similar to that of erythromycin. By inhibiting the protein synthesis process of pathogens, it has high activity against Gram-positive bacteria and some Gram-negative bacteria, including Staphylococcus aureus, hemolytic streptococcus, gonococcus, diplococcus meningitidis, Bordetella pertussis, mycoplasma, chlamydia, etc. It is also effective against spirochetes, rickettsiae and large viruses. Clinically, it is used for respiratory tract infections, bronchitis, pneumonia, paranasal sinusitis, otitis media, skin infections, postoperative infections, urology, otolaryngology, dental infections, etc., and can also be used for infections caused by staphylococci resistant to penicillin and erythromycin.

[0003] Currently, the production of josamycin mainly comes from imported raw materials. The domestic production level of josamycin is low and the cost is high, and there is no manufacturer producing raw materials. Therefore, improving the fermentation production capacity of josamycin and realizing industrialized production in China is of great significance. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a high-efficiency and stable medium for fermenting and producing josamycin and a method thereof. The method provided by the present invention can be used for industrial scale production.

[0005] Solution for Solving the Problem

[0006] On the one hand, the present invention provides a fermentation medium for fermenting and preparing josamycin. The fermentation medium contains a carbon source and a nitrogen source. The carbon source is selected from one or more of soybean oil, peanut oil, rapeseed oil, and olive oil; the nitrogen source is selected from one or more of corn gluten meal, sesame cake powder, soybean cake powder, cottonseed cake powder, biological nitrogen, and L-leucine.

[0007] Preferably, the carbon source is selected from soybean oil and / or olive oil; the nitrogen source is selected from one or more of corn gluten meal, sesame cake powder, biological nitrogen, and L-leucine.

[0008] Preferably, the mass-volume percentage content of the carbon source is 6-10%, preferably 7-10%, more preferably 7-9%, and most preferably 8%.

[0009] Preferably, the mass-volume percentage content of the nitrogen source is 3-6%, preferably 3.5-5%, and more preferably 4-5%.

[0010] Further preferably, the nitrogen source consists of corn gluten meal, sesame cake powder and L-leucine, or the nitrogen source consists of corn gluten meal and biotin.

[0011] More preferably, the mass-volume percentage content of the corn gluten meal is 2% to 3%, preferably 2 to 2.6%.

[0012] More preferably, the mass-volume percentage content of the sesame cake powder is 0.5% to 2%, preferably 1 to 1.6%.

[0013] More preferably, the mass-volume percentage content of the L-leucine is 0.5% to 1.0%, preferably 0.5 to 0.8%.

[0014] Further preferably, the mass-volume percentage content of the biotin is 1 to 3%, preferably 1 to 2%.

[0015] Preferably, the fermentation medium further comprises inorganic salts, and the inorganic salts are selected from one or more of ferrous sulfate, manganese sulfate, magnesium sulfate, sodium glutamate, and calcium carbonate.

[0016] Preferably, the mass-volume ratio content of the inorganic salts is 0.3 to 0.7%, preferably 0.35 to 0.55%, more preferably 0.39 to 0.51%.

[0017] Further preferably, the mass-volume percentage content of the ferrous sulfate is 0.01% to 0.05%, preferably 0.02 to 0.05%, more preferably 0.02 to 0.04%, and most preferably 0.03%.

[0018] Further preferably, the mass-volume percentage content of the manganese sulfate is 0.01% to 0.05%, preferably 0.02 to 0.05%, more preferably 0.02 to 0.04%, and most preferably 0.03%.

[0019] Further preferably, the mass-volume percentage content of the magnesium sulfate is 0.01% to 0.05%, preferably 0.02 to 0.05%, more preferably 0.02 to 0.04%, and most preferably 0.03%.

[0020] Further preferably, the mass-volume percentage content of the calcium carbonate is 0.2% to 0.5%, preferably 0.25 to 0.5%, more preferably 0.25 to 0.4%.

[0021] Further preferably, the mass-volume percentage content of the sodium glutamate is 0.05% to 0.15%, preferably 0.10 to 0.15%, more preferably 0.12%.

[0022] Preferably, the fermentation medium further comprises polypropylene glycol, and the mass - volume percentage content of the polypropylene glycol is 0.01 - 0.02%.

[0023] Preferably, the components of the fermentation medium and their mass - volume percentage contents are as follows:

[0024]

[0025] Or,

[0026]

[0027]

[0028] On the other hand, the present invention provides a method for preparing josamycin by fermentation, and the above - mentioned fermentation medium is used for fermentation.

[0029] Preferably, the method comprises the following steps:

[0030] (1) Inoculate the seed liquid into the fermentation medium described in any one of the above to carry out fermentation culture;

[0031] (2) Isolate and obtain josamycin from the fermentation broth.

[0032] Preferably, in step (1), control the temperature of the fermentation culture to be 29 - 31°C; and / or, control the dissolved oxygen in the fermentation system to be ≥20%.

[0033] Preferably, the strain used for fermentation is Streptomyces narbonensis or its mutagenized strain, preferably Streptomyces narbonensis ATCC 17835.

[0034] Effects of the Invention

[0035] The present invention provides a fermentation medium for fermentative production of josamycin using soybean oil, peanut oil, rapeseed oil, olive oil, etc. as carbon sources and corn gluten meal, sesame cake powder, soybean cake powder, cottonseed cake powder, biological nitrogen, L - leucine, etc. as nitrogen sources. Using the fermentation medium provided by the present invention improves the biosynthesis efficiency of josamycin during the fermentation process, increases the fermentation yield, reduces the cost, and is suitable for industrial production. Detailed Embodiments

[0036] To make the technical solutions and beneficial effects of the present invention more obvious and understandable, the following provides a detailed description by way of listing specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following examples are generally carried out according to conventional experimental conditions. The reagents and raw materials used in the present invention are commercially available unless otherwise specified.

[0037] Josamycin, a macrolide antibiotic produced by fermentation of Streptomyces narbonensis, was discovered by Professor Umezawa Hiroo of the Institute of Microbial Chemistry in Japan. It was developed and launched by Fujisawa (now Astellas Pharma) in Japan in 1970. However, aiming at the problem of low production level, the inventors found through extensive and in-depth research that the production strain can improve the yield of josamycin by fermenting and culturing with a culture medium containing one or more of soybean oil, peanut oil, rapeseed oil, and olive oil with a certain mass-volume fraction. Especially when soybean oil is used as the carbon source, the production cost can be significantly reduced. Based on this, the inventors completed the present invention.

[0038] The mass-volume percentage (%) in the present invention means that there are several grams of solute in every 100 mL of solution.

[0039] In the present invention, the fermentation strain refers to any strain applicable to fermentative production of josamycin in the prior art, such as Streptomyces narbonensis or its mutagenized strain. The strain used in the fermentation of the examples of the present invention is Streptomyces narbonensis ATCC 17835, which is derived from the American Type Culture Collection (ATCC).

[0040] On the one hand, the present invention provides a fermentation medium for fermentatively preparing josamycin, the fermentation medium contains a carbon source and a nitrogen source, the carbon source is selected from one or more of soybean oil, peanut oil, rapeseed oil, and olive oil; the nitrogen source is selected from one or more of corn gluten meal, sesame cake meal, soybean cake meal, cottonseed cake meal, bio-nitrogen, and L-leucine.

[0041] In some embodiments, the carbon source is selected from soybean oil and / or olive oil.

[0042] In some embodiments, the mass-volume percentage content of the carbon source is 6-10%, such as 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc.

[0043] In some embodiments, the mass-volume percentage content of the carbon source is 7-10%.

[0044] In some embodiments, the mass-volume percentage content of the carbon source is 7-9%.

[0045] In some embodiments, the mass-volume percentage content of the carbon source is 8%.

[0046] In some embodiments, the nitrogen source is selected from one or more of corn gluten meal, sesame cake meal, bio-nitrogen, and L-leucine.

[0047] In some embodiments, the mass - volume percentage content of the nitrogen source is 3 - 7%, such as 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, etc.

[0048] In some embodiments, the mass - volume percentage content of the nitrogen source is 3.5 - 5%.

[0049] In some embodiments, the mass - volume percentage content of the nitrogen source is 4 - 5%.

[0050] In some embodiments, the nitrogen source is composed of corn gluten meal, sesame cake meal and L - leucine.

[0051] In some embodiments, the nitrogen source is composed of corn gluten meal and biotin.

[0052] In some embodiments, the mass - volume percentage content of the corn gluten meal is 2% - 4%, such as 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, etc.

[0053] In some embodiments, the mass - volume percentage content of the corn gluten meal is 2.2 - 3.8%.

[0054] In some embodiments, the mass - volume percentage content of the corn gluten meal is 2.4 - 3.6%.

[0055] In some embodiments, the mass - volume percentage content of the sesame cake meal is 0.5% - 2%, such as 1%, 1.2%, 1.4%, 1.4%, 1.6%, 1.8%, 2%, etc.

[0056] In some embodiments, the mass - volume percentage content of the sesame cake meal is 1 - 1.6%.

[0057] In some embodiments, the mass - volume percentage content of the L - leucine is 0.5% - 1.0%, such as 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, etc.

[0058] In some embodiments, the mass - volume percentage content of the L - leucine is 0.5 - 0.8%.

[0059] In some embodiments, the mass - volume percentage content of the L - leucine is 0.6%.

[0060] In some embodiments, the mass - volume percentage content of the biological nitrogen is 1 - 3%, such as 1.1%, 1.2%, 1.3%, 1.4, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, etc.

[0061] In some embodiments, the mass - volume percentage content of the biological nitrogen is 1 - 2%.

[0062] In some embodiments, the mass - volume percentage content of the biological nitrogen is 1 - 1.5%.

[0063] In some embodiments, the mass - volume percentage content of the biological nitrogen is 1.2%.

[0064] In some embodiments, the fermentation medium further comprises inorganic salts.

[0065] In some embodiments, the inorganic salts are selected from one or more of ferrous sulfate, manganese sulfate, magnesium sulfate, sodium glutamate, and calcium carbonate.

[0066] In some embodiments, the mass - volume ratio content of the inorganic salts is 0.28 - 0.8%, such as 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.8%, etc.

[0067] In some embodiments, the mass - volume ratio content of the inorganic salts is 0.35 - 0.55%.

[0068] In some embodiments, the mass - volume ratio content of the inorganic salts is 0.39 - 0.51%.

[0069] In some embodiments, the mass - volume percentage content of ferrous sulfate is 0.01 - 0.05%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, etc.

[0070] In some embodiments, the mass - volume percentage content of ferrous sulfate is 0.02 - 0.05%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, etc.

[0071] In some embodiments, the mass - volume percentage content of ferrous sulfate is 0.02 - 0.04%.

[0072] In some embodiments, the mass - volume percentage content of ferrous sulfate is 0.03%.

[0073] In some embodiments, the mass - volume percentage content of manganese sulfate is 0.01% - 0.05%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, etc.

[0074] In some embodiments, the mass - volume percentage content of manganese sulfate is 0.02 - 0.05%.

[0075] In some embodiments, the mass - volume percentage content of manganese sulfate is 0.02 - 0.04%.

[0076] In some embodiments, the mass - volume percentage content of manganese sulfate is 0.03%.

[0077] In some embodiments, the mass - volume percentage content of magnesium sulfate is 0.01% - 0.05%, such as 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, etc.

[0078] In some embodiments, the mass - volume percentage content of magnesium sulfate is 0.02 - 0.05%.

[0079] In some embodiments, the mass - volume percentage content of magnesium sulfate is 0.02 - 0.04%.

[0080] In some embodiments, the mass - volume percentage content of magnesium sulfate is 0.03%.

[0081] In some embodiments, the mass - volume percentage content of calcium carbonate is 0.2% - 0.5%, such as 0.2%, 0.3%, 0.4%, 0.5%, etc.

[0082] In some embodiments, the mass - volume percentage content of calcium carbonate is 0.25 - 0.5%.

[0083] In some embodiments, the mass - volume percentage content of calcium carbonate is 0.25 - 0.4%.

[0084] In some embodiments, the mass - volume percentage content of calcium carbonate is 0.3%.

[0085] In some embodiments, the mass - volume percentage content of sodium glutamate is 0.05% - 0.15%, such as 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, etc.

[0086] In some embodiments, the mass - volume percentage content of sodium glutamate is 0.10% - 0.15%.

[0087] In some embodiments, the mass - volume percentage content of sodium glutamate is 0.12%.

[0088] In some embodiments, the fermentation medium further comprises polypropylene glycol, and the mass - volume percentage content of the polypropylene glycol is 0.01% - 0.02%, such as 0.012%, 0.014%, 0.016%, 0.018%, 0.02%, etc.

[0089] In the present invention, there is no limitation on the polypropylene glycol. In some embodiments, the polypropylene glycol includes but is not limited to polypropylene glycol 2000, polypropylene glycol 4000, polypropylene glycol 6000, polypropylene glycol 8000, etc.

[0090] In some embodiments, the polypropylene glycol is polypropylene glycol 2000.

[0091] In some embodiments, the components of the fermentation medium and their mass - volume percentage contents are as follows,

[0092]

[0093] In some embodiments, the components of the fermentation medium and their mass - volume percentage contents are as follows,

[0094]

[0095]

[0096] On the other hand, the present invention provides a method for preparing josamycin by fermentation, using the above - mentioned fermentation medium.

[0097] In some embodiments, the method comprises the following steps:

[0098] (1) Inoculating the seed liquid into the fermentation medium described in any one of the above to carry out fermentation culture;

[0099] (2) Separating josamycin from the fermentation broth.

[0100] In some embodiments, the method includes culturing the fermentation broth at 29 - 31 °C.

[0101] In some embodiments, the method includes controlling the fermentation culture time to be 6 - 7 days.

[0102] In some embodiments, the method includes controlling the fermentation culture time to be 4 - 5 days.

[0103] In some embodiments, controlling the dissolved oxygen in the fermentation system ≥20%.

[0104] In some embodiments, the strain used for fermentation is Streptomyces narbonensis or its mutagenized strain.

[0105] In some embodiments, the strain used for fermentation is Streptomyces narbonensis ATCC 17835.

[0106] In some embodiments, the production strain is inoculated into a seed medium for cultivation to obtain a seed liquid.

[0107] In some embodiments, the seed medium contains soybean cake powder, yeast extract powder, dry corn steep liquor, corn protein powder, soybean oil, and calcium carbonate.

[0108] In some embodiments, the components in the seed medium may be that the mass - volume percentage content of soybean cake powder is 0.3% - 0.8%, the mass - volume percentage content of yeast extract powder is 0.3% - 0.8%, the mass - volume percentage content of dry corn steep liquor is 0.3% - 0.8%, the mass - volume percentage content of corn protein powder is 0.3% - 0.8%, the mass - volume percentage content of soybean oil is 0.3% - 0.8%, and the content of calcium carbonate is 0.2% - 0.5%.

[0109] In some embodiments, the components in the seed medium may be that the mass - volume percentage content of soybean cake powder is 0.5%, the mass - volume percentage content of yeast extract powder is 0.5%, the mass - volume percentage content of dry corn steep liquor is 0.5%, the mass - volume percentage content of corn protein powder is 0.5%, the mass - volume percentage content of soybean oil is 0.5%, and the mass - volume percentage content of calcium carbonate is 0.3%.

[0110] In some embodiments, the seed medium is adjusted with conventional acids and bases to make its pH 6.7 - 6.9 before sterilization.

[0111] In some embodiments, the pH of the seed medium is 6.8.

[0112] The present invention will be further described below through specific examples. Unless otherwise specified herein, "%" represents mass - volume percentage. The materials and reagents in the following examples, unless otherwise specified, are commonly used materials or reagents in the art, and can all be obtained commercially or synthesized by known methods. The experimental methods without specified conditions in the following examples are usually carried out according to conventional experimental conditions or the conditions recommended by the manufacturers of relevant reagents (kits).

[0113] Example 1: 50L Tank Fermentation Production of Josamycin

[0114] Prepare the seed medium. The components of the medium are as follows: soybean cake powder 0.5%, yeast extract powder 0.5%, corn steep liquor dry powder 0.5%, corn protein powder 0.5%, soybean oil 0.5%, calcium carbonate 0.3%. Prepare with drinking water and adjust the pH to 6.8 before sterilization. Sterilize at 121 °C for 30 minutes.

[0115] First-stage shake flask seed culture: Inoculate 0.5 mL of the seed glycerol tube into the first-stage seed shake flask, place it on a shaker at 30 °C, 250 rpm, and culture for 24 hours.

[0116] Second-stage shake flask seed culture: Inoculate 3 mL of the first-stage shake flask seed into the second-stage seed shake flask, place it on a shaker at 30 °C, 250 rpm, and culture for 24 hours.

[0117] Prepare a fermentation medium with a volume of 35 L. The components of the medium are as follows: soybean oil 8%, corn protein powder 3.6%, bio nitrogen 1.2%, sodium glutamate 0.12%, ferrous sulfate 0.03%, manganese sulfate 0.03%, magnesium sulfate 0.03%, calcium carbonate 0.3%. Make up the volume to 35 L with drinking water and adjust the pH to 7.5 before sterilization. Sterilize at 121 °C for 30 minutes.

[0118] 50 L fermenter culture: Inoculate the second-stage shake flask seed liquid into the fermenter, and the inoculation amount is 5% (V / V%) of the volume of the fermentation broth. The culture temperature is 30 °C, control the stirring speed at 200 - 500 RPM, the air flow rate is 0.5 - 1 VVM, the tank pressure is 0.05 Mpa, monitor the dissolved oxygen electrode online to make the dissolved oxygen control ≥ 20% (the initial dissolved oxygen calibration of the fermenter culture is 100%). If the dissolved oxygen is lower than 20%, then flow and supplement water at 0.1% / h - 0.2% / h. If the offline pH is lower than 6.4 during the fermentation process, then supplement ammonia water or 20% sodium hydroxide to control the offline pH at 6.5 - 7.0. Supplement carbon source: If the carbon source content is lower than 3.0% from 72 h to 120 h, then flow and supplement at 0.05% / h - 0.1% / h to maintain 2.5% - 3.5%. Fermentation culture for 6 - 7 days.

[0119] During the fermentation process, sample to measure the content of josamycin in the fermentation broth. The treatment process is as follows: Take 2 ml of the fermentation broth, place it in a 50 mL centrifuge tube, add 18 mL of absolute ethanol, mix well, ultrasonicate for 30 minutes, shake well, take an appropriate amount of the sample solution, place it in a high-speed centrifuge, centrifuge at 20000 revolutions per minute for 1 minute, take out, take the supernatant and filter it with a 0.22 μm filter membrane. The filtrate is used as the test sample solution, and HPLC is used to measure its yield. The result shows that the content of josamycin in the fermentation broth is 7152 mg / L.

[0120] Examples 2 - 5: Production of josamycin with different fermentation media

[0121] Josamycin was prepared by the fermentation method of Example 1 using fermentation media with different compositions. The inorganic salt components in the fermentation medium were: ferrous sulfate 0.03%, manganese sulfate 0.03%, magnesium sulfate 0.03%, and calcium carbonate 0.3%. The carbon source, nitrogen source, and other components of the fermentation medium are shown in Table 1.

[0122] Table 1

[0123]

[0124] The josamycin content in the fermentation broth obtained in Examples 2 to 5 was determined by the detection method of Example 1, as shown in Table 2.

[0125] Table 2

[0126]

[0127] Example 6: 20T Fermentation Production of Josamycin

[0128] The first-stage shake flask seed culture and the second-stage shake flask seed culture were the same as in Example 1.

[0129] Prepare the first-stage seed tank medium, and the medium components were as follows: soybean cake powder 0.5%, yeast extract powder 0.5%, corn steep liquor dry powder 0.5%, corn protein powder 0.5%, soybean oil 0.5%, and calcium carbonate 0.3%. Adjust the pH to 6.8 before sterilization. Sterilize online with steam at 121°C for 30 minutes.

[0130] First-stage seed tank culture: The second-stage shake flask seed liquid was inoculated into the first-stage seed tank at an inoculation amount of 1% - 2% by volume. The culture temperature was 30°C, and the pH and dissolved oxygen were not controlled. Cultivate for 1 day.

[0131] Prepare the second-stage seed tank medium, and the preparation method was the same as that of the first-stage seed tank.

[0132] Second-stage seed tank culture: The first-stage seed tank seed liquid was inoculated into the second-stage seed tank at an inoculation amount of 2% - 5% by volume. The culture temperature was 30°C, and the pH and dissolved oxygen were not controlled. Cultivate for 1 day.

[0133] Prepare the 20T fermenter medium, and the medium components were as follows: soybean oil 8%, corn protein powder 2.4%, sesame cake powder 1.2%, L-leucine 0.6%, ferrous sulfate 0.03%, manganese sulfate 0.03%, magnesium sulfate 0.03%, calcium carbonate 0.3%, and make up to 14T with drinking water. Adjust the pH to 7.5 before sterilization. Sterilize online with steam at 121°C for 30 minutes.

[0134] 20T fermentor culture: The seed liquid from the secondary seed tank is transferred to the 20T fermentor at a volume ratio of 5-10%. The culture temperature is 30°C, and the dissolved oxygen is linked to the rotation speed, with the dissolved oxygen controlled at ≥20%. pH: If the off-line pH is lower than 6.4, ammonia water or 20% sodium hydroxide is added to control the off-line pH at 6.5-7.0. Water addition: If the dissolved oxygen is lower than 20%, water is fed at a rate of 0.1% / h - 0.2% / h. Carbon source supplementation: If the carbon source content is lower than 3.0% from 72h to 120h, it is fed at a rate of 0.05% / h - 0.1% / h to maintain it at 2.5% - 3.5%.

[0135] After fermentation for a certain period of time, the content of josamycin in the fermentor was determined by the detection method of Example 1. The yield changes of josamycin fermented in this example are shown in Table 3 below.

[0136] Table 3

[0137]

[0138]

[0139] As can be seen from the above table, when cultured in a 20T fermentor for 4-5 days, the yield can reach over 7 g / L, and the results of three batches of fermentation experiments have good reproducibility. The fermentation method of josamycin provided by the present invention is efficient, stable, and low-cost, and can be applied to industrial production.

[0140] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various modifications and changes can be made based on the above embodiments. Similarly, any combination of the technical features of the above embodiments can be made to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. A fermentation medium for fermentatively preparing josamycin, characterized in that, The fermentation medium contains a carbon source and a nitrogen source. The carbon source is selected from one or more of soybean oil, peanut oil, rapeseed oil, and olive oil; the nitrogen source is selected from one or more of corn protein powder, sesame cake powder, soybean cake powder, cottonseed cake powder, biological nitrogen, and L-leucine.

2. The fermentation medium according to claim 1, wherein The carbon source is selected from soybean oil and / or olive oil; the nitrogen source is selected from one or more of corn protein powder, sesame cake powder, biological nitrogen, and L-leucine.

3. The fermentation medium according to claim 1, characterized in that, The mass-volume percentage content of the carbon source is 6-10%, preferably 7-10%, more preferably 7-9%, and most preferably 8%; Preferably, the mass-volume percentage content of the nitrogen source is 3-7%, preferably 3.5-5%, and more preferably 4-5%.

4. The fermentation medium according to claim 1, wherein The nitrogen source consists of corn protein powder, sesame cake powder, and L-leucine or the nitrogen source consists of corn protein powder and biological nitrogen; Preferably, the mass-volume percentage content of the corn protein powder is 2%-4%, preferably 2.2-3.8%; Preferably, the mass-volume percentage content of the sesame cake powder is 0.5%-2%, preferably 1-1.6%; Preferably, the mass-volume percentage content of L-leucine is 0.5%-1.0%, preferably 0.5-0.8%; Preferably, the mass-volume percentage content of biological nitrogen is 1-3%, preferably 1-2%.

5. The fermentation medium according to claim 1, wherein The fermentation medium further contains inorganic salts, and the inorganic salts are selected from one or more of ferrous sulfate, manganese sulfate, magnesium sulfate, sodium glutamate, and calcium carbonate; Preferably, the mass-volume ratio content of the inorganic salts is 0.28-0.8%, preferably 0.35-0.55%, and more preferably 0.39-0.51%.

6. The fermentation medium according to claim 5, characterized in that, The mass-volume percentage content of ferrous sulfate is 0.01%-0.05%, preferably 0.02-0.05%, more preferably 0.02-0.04%, and most preferably 0.03%; Preferably, the mass-volume percentage content of manganese sulfate is 0.01%-0.05%, preferably 0.02-0.05%, more preferably 0.02-0.04%, and most preferably 0.03%; Preferably, the mass-volume percentage content of magnesium sulfate is 0.01%-0.05%, preferably 0.02-0.05%, more preferably 0.02-0.04%, and most preferably 0.03%; Preferably, the mass-volume percentage content of calcium carbonate is 0.2%-0.5%, preferably 0.25-0.5%, more preferably 0.25-0.4%; most preferably 0.3%; Preferably, the mass-volume percentage content of sodium glutamate is 0.05%-0.15%, preferably 0.10-0.15%, and more preferably 0.12%.

7. The fermentation medium according to any one of claims 1 to 6, characterized in that, The fermentation medium also includes polypropylene glycol, and the mass-volume percentage content of the polypropylene glycol is 0.01-0.02%.

8. The fermentation medium according to claim 1, wherein, The components of the fermentation medium and their mass-volume percentage contents are: The balance is water; Or, The balance is water.

9. A method for preparing josamycin by fermentation method, characterized in that, Use the fermentation medium according to any one of claims 1-8 for fermentation.

10. The method according to claim 9, wherein The method includes the following steps: (1) Inoculate the seed liquid into the fermentation medium according to any one of claims 1 to 8 for fermentation culture; (2) Isolate josamycin from the fermentation broth.

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