A fermentation method for continuously producing erythromycin by using bacterial fermentation liquid

Abstract

The present invention is a fermentation method for continuously producing erythromycin by using bacterial contamination fermentation liquid, wherein the method uses erythromycin-resistant Saccharopolyspora as production bacteria, and adds antibiotics corresponding to the acquired resistance into the bacterial contamination fermentation liquid. The present invention successfully solves the problem of inactivating the culture medium due to bacterial contamination in the erythromycin production process, and effectively improves the technical level of erythromycin fermentation process.

Description

Technical Field

[0001] The invention belongs to the technical field of industrial microbial fermentation, and in particular relates to a fermentation method for continuously producing erythromycin by utilizing contaminated fermentation liquid. Background Art

[0002] There are many reasons for contamination during erythromycin fermentation, mainly including contamination in seeds, contamination in sterile air, incomplete sterilization of culture medium and equipment, dead corners and leakage of equipment, operational errors and poor technical management.

[0003] During the production process, sterility testing is usually carried out through inspections of biochemical indicators and microscopic observations, fermentation equipment is periodically inspected and maintained, and the operation process is strictly carried out as required to prevent contamination by foreign bacteria.

[0004] Although strict prevention measures are taken in every link of the fermentation process, bacterial contamination incidents still occur. There is nothing we can do about the contaminated fermentation liquid, and direct inactivation treatment causes serious economic losses.

[0005] At present, fermentation factories focus on the selection of strains with strong resistance to foreign bacteria contamination, culture medium sterilization methods, hygiene and on-site management, periodic inspection and maintenance and other pollution prevention and control aspects. There is no systematic research on the impact of contaminating bacteria on fermentation, which leads to great blindness in the prevention and control of foreign bacteria contamination.

[0006] However, the inventors of this application found that the above technology has at least the following technical problems in the process of implementing the technical solution of the invention in the embodiment of this application:

[0007] In the industrial fermentation of erythromycin, bacterial contamination is a very serious problem in production. Usually, the fermentation liquid is inactivated, which not only increases the fermentation cost, but also affects the benefits of refining and purification, resulting in low product yield, reduced product quality, and economic losses. Summary of the invention

[0008] The invention aims to solve the problem that the fermentation process of erythromycin cannot be continued due to contamination of foreign bacteria. By adding sterile gentamicin aqueous solution and ampicillin aqueous solution into the fermentation liquid, the foreign bacteria are killed and the growth of the production bacteria is blocked.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is:

[0010] In some embodiments of the present invention, a fermentation method for continuously producing erythromycin using a bacterial fermentation broth is provided, wherein erythromycin-resistant Saccharopolyspora erythrae is used as the production bacterium, and an antibiotic corresponding to the resistance is added to the bacterial fermentation broth.

[0011] Furthermore, the resistant Saccharopolyspora erythraea is prepared by a corresponding antibiotic resistance screening method.

[0012] Furthermore, the production bacteria is Saccharopolyspora erythraea with gentamicin resistance, and the antibiotic is gentamicin.

[0013] Furthermore, the gentamicin has an inhibitory concentration of 3-12 μg / ml.

[0014] Furthermore, the bacterial contaminant is selected from at least one of long bacillus, short bacillus and small bacillus.

[0015] Furthermore, the production bacteria is Saccharopolyspora erythraea with ampicillin resistance, and the antibiotic is ampicillin.

[0016] Furthermore, the concentration of ampicillin that inhibits bacterial infection is 3-12 μg / ml.

[0017] Furthermore, the bacterial contaminant is selected from at least one of long bacillus, short bacillus and small bacillus.

[0018] The operations used in the fermentation method for continuously producing erythromycin using the contaminated fermentation broth are all routine operations unless otherwise specified.

[0019] Unless otherwise specified, the raw materials and reagents used in the experiments of the present invention can be purchased from regular suppliers.

[0020] The erythromycin producing bacteria used in the experiment of the present invention can grow normally in a fermentation medium mixed with gentamicin at a concentration of 0-12 μg / ml and ampicillin at a concentration of 0-12 μg / ml.

[0021] The volume of the erythromycin fermentation tank in the experiment of the present invention is 186m 3 , fermentation liquid volume 130m 3 The fermentation medium composition is: 5% starch, 8% soybean meal, 3% dextrin, 0.2% (NH4)2SO4, 0.5% CaCO3. After 24 hours of fermentation, 1% n-propanol is added.

[0022] The culture medium in the experiment of the present invention was sterilized by high pressure steam sterilization at a temperature of 125° C. for 30 min.

[0023] During the experiment of the present invention, when the fermentation broth was found to be contaminated with bacteria by microscopic examination, 0.39kg-1.56kg of gentamicin and 0.39kg-1.56kg of ampicillin were weighed and prepared into an aqueous solution, and a 0.22μm membrane filtration device was used, and the filtration rate was maintained at 1.0m 3 / h, filter and sterilize. Use the pressure difference method to quickly add it to the fermentation tank, start stirring, let in air, and mix thoroughly.

[0024] During the experiment of the present invention, after the gentamicin and ampicillin aqueous solution was added for one hour, samples were taken at regular intervals for microscopic examination, and the number of bacteria in the field of view was observed under a 10x100x microscope. When the number no longer increased, it indicated that the bacteria were suppressed.

[0025] In the experiment of the present invention, the amount of gentamicin and ampicillin added to the fermentation tank shall not be less than 0.39 kg and shall not be higher than 1.56 kg.

[0026] The fermentation cycle in the experimental process of the present invention is 150h.

[0027] The technical solution of the present invention has the following beneficial technical effects:

[0028] The invention achieves the technical effect of inhibiting the influence of miscellaneous bacteria on normal production bacteria and continuing the erythromycin fermentation process by adding antibiotics corresponding to the resistance to the contaminated fermentation liquid, and avoids inactivation of the fermentation liquid due to contamination. DETAILED DESCRIPTION

[0029] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0030] Experimental Example 1 Preparation of a strain producing dual resistance to gentamicin and ampicillin

[0031] Using ribosome engineering technology, gentamicin and ampicillin were combined to screen the resistance of Saccharopolyspora erythraea. A multi-round progressive resistance screening method was adopted, and a total of 10 rounds of screening were performed. The shake flask titer of the final dual-resistance production strain was 19.3% higher than that of the original strain. The resistance concentrations of gentamicin and ampicillin obtained by this strain were 13μg / ml.

[0032] The preparation method is as follows:

[0033] First round: The minimum inhibitory concentration (MIC) of gentamicin for the starting strain is 3.2μg / ml, and the minimum inhibitory concentration (MIC) of ampicillin for the starting strain is 5.4μg / ml. The two antibiotics were mixed in equal proportions, and the starting concentration of 4μg / ml was selected for the first round of screening. The original production strain was used as the starting strain to prepare a spore suspension, and the concentration was controlled at 10 8 / ml, and then diluted to 100 times, 1000 times, and 10000 times, respectively, and then spread on a plate containing a mixture of gentamicin and ampicillin at 4μg / ml, cultured for 7 days, and single colonies were picked to prepare slant strains for verification. The strain with the highest titer was retained for the next round of resistance screening.

[0034] Second round: Increase the resistance screening concentration to 5 μg / ml, select the resistant strain with the highest titer in the first round to prepare spore suspension, and the screening method is the same as the first round.

[0035] The same method was followed, with the concentration of each round of resistance screening increasing by 1 μg / ml compared to the previous round, and 10 rounds of screening were performed continuously.

[0036] Experimental Example 2 Investigation of the sensitivity of the dual-resistant strain to a mixture of gentamicin and ampicillin in fermentation broth

[0037] ① Prepare the culture medium. Set the concentration gradient of gentamicin and ampicillin mixture to 9.0μg / ml, 10.0μg / ml, 11.0μg / ml, 12.0μg / ml, 13.0μg / ml, 14.0μg / ml, 15.0μg / ml, 16.0μg / ml. Prepare erythromycin fermentation medium, take 30ml and dispense it into 300ml Erlenmeyer flasks, three parallel samples for each gradient, sterilize with high-pressure steam for 30min, wait until the culture medium temperature drops to room temperature before use, and add sterile ampicillin and gentamicin aqueous solutions of corresponding concentrations into the Erlenmeyer flasks according to the above concentration gradient.

[0038] ② Inoculation and culture. Take out the erythromycin-producing bacteria slant with resistance to gentamicin and ampicillin, inoculate a spatula of spores in the seed bottle, and after culturing for 24 hours, inoculate it into the prepared triangular flask according to the inoculation amount of 15%, and culture it for 48 hours. Observe the growth of the producing bacteria under a 10x100x microscope.

[0039] ③ The results show that when the concentration of ampicillin and gentamicin in the fermentation medium is 15.0 μg / ml, the growth of erythromycin-producing bacteria is inhibited, and when the concentration is greater than 18.0 μg / ml, the growth of erythromycin-producing bacteria is significantly inhibited. Therefore, the concentration of the two antibiotics added to the fermentation broth in the experiment of the present invention shall not be higher than 12.0 μg / ml.

[0040] Test Example 3 Isolation of Miscellaneous Bacteria in Erythromycin Fermentation Broth

[0041] According to statistics, the bacteria infected in the production process of erythromycin fermentation in our company include long bacillus, short bacillus, and small bacillus. The fermentation liquid after infection is sampled by aseptic operation, 100 μL is spread on solid LB medium for separation and purification, and a single colony is picked to prepare a test tube slant for storage. The test tube slant strain is separated and purified again, and a single colony is picked to prepare a test tube slant for standby use.

[0042] Test Example 4 Investigation of the sensitivity of miscellaneous bacteria to gentamicin

[0043] ① Prepare the culture medium. Set the concentration gradient of gentamicin to 0.0μg / ml, 0.3μg / ml, 0.6μg / ml, 0.9μg / ml, 1.2μg / ml, 1.5μg / ml, 1.8μg / ml, 2.1μg / ml, 2.4μg / ml. Prepare erythromycin fermentation medium, take 30ml and dispense it into 300ml Erlenmeyer flasks, three parallels for each gradient, sterilize with high-pressure steam for 30min, wait until the temperature of the culture medium drops to room temperature before use, and add sterile gentamicin aqueous solution of corresponding concentration into the Erlenmeyer flask according to the above concentration gradient.

[0044] ②Inoculation and culture. Take out the test tube slopes of three kinds of bacteria and conduct tests respectively. Inoculate a ring of bacteria in a triangular flask, culture overnight, and observe the growth of bacteria. Under normal circumstances, erythromycin-producing bacteria grow in this culture medium, and the culture medium is relatively viscous. When bacteria grow in this culture medium, the culture medium will become very thin. Therefore, the growth of bacteria can be judged according to the state of the culture medium.

[0045] ③ Record the minimum inhibitory concentration. The minimum inhibitory concentration was recorded based on the comprehensive situation of the three parallel samples. The results are shown in the following table. When the concentration of gentamicin in the fermentation broth reaches 1.8 μg / ml, it can completely inhibit the growth of bacteria and kill them.

[0046]

[0047] Note: “+” represents thin fermentation liquid; “-” represents thick fermentation liquid

[0048] Test Example 5 Investigation of the sensitivity of miscellaneous bacteria to ampicillin

[0049] ① Prepare the culture medium. Set the concentration gradient of ampicillin to 0.0μg / ml, 0.4μg / ml, 0.8μg / ml, 1.2μg / ml, 1.6μg / ml, 2.0μg / ml, 2.4μg / ml, 2.8μg / ml, 3.2μg / ml. Prepare erythromycin fermentation medium, take 30ml and divide it into 300ml Erlenmeyer flasks, three parallels for each gradient, sterilize with high-pressure steam for 30min, wait until the temperature of the culture medium drops to room temperature before use, and add sterile ampicillin aqueous solution of corresponding concentration into the Erlenmeyer flask according to the above concentration gradient.

[0050] ②Inoculation and culture. Take out the test tube slopes of three kinds of bacteria and conduct tests respectively. Inoculate a ring of bacteria in a triangular flask, culture overnight, and observe the growth of bacteria. Under normal circumstances, erythromycin-producing bacteria grow in this culture medium, and the culture medium is relatively viscous. When bacteria grow in this culture medium, the culture medium will become very thin. Therefore, the growth of bacteria can be judged according to the state of the culture medium.

[0051] ③ Record the minimum inhibitory concentration. The minimum inhibitory concentration was recorded based on the comprehensive situation of the three parallel samples. The results are shown in the following table. When the concentration of ampicillin in the fermentation broth reaches 2.8 μg / ml, the growth of bacteria can be completely inhibited and the bacteria can be killed.

[0052]

[0053] Test Example 6 Determination of the antibacterial concentration of gentamicin and ampicillin

[0054] According to the experimental results, the minimum inhibitory concentration of gentamicin against miscellaneous bacteria is 1.8 μg / ml, and the minimum inhibitory concentration of ampicillin against miscellaneous bacteria is 2.8 μg / ml. In order to achieve a better antibacterial effect, the minimum inhibitory concentrations of the two antibiotics are calculated according to 3.0 μg / ml. Therefore, the concentration of each antibiotic added to the fermentation broth in the experiment of the present invention shall not be less than 3.0 μg / ml.

[0055] Example 1

[0056] 1) During the erythromycin fermentation process of the example, long-stained bacilli were found under microscopic examination at 48h of the fermentation cycle, and 3 strains were observed under the field of view of a 10x100x microscope.

[0057] 2) Prepare an aqueous solution containing 0.5 kg of gentamicin and 0.5 kg of ampicillin, filter and sterilize through a 0.22 μm filter membrane, and then feed into the fermenter.

[0058] 3) Samples were taken for microscopic examination at 50h, 52h, 54h, and 56h of the fermentation cycle, and 6, 9, 5, and 6 strains of long bacilli were observed under the field of view of 10x100 magnification microscope. The number no longer increased, indicating that the growth of long bacilli was completely inhibited.

[0059] 4) The fermentation was terminated after 150 h, and the erythromycin A titer was detected to be 7635 u / ml.

[0060] Example 2

[0061] 1) During the erythromycin fermentation process of the example, stained brevis bacteria were found under microscopic examination at 92 hours of the fermentation cycle, and 5 strains were observed under the field of view of a 10x100x microscope.

[0062] 2) Prepare an aqueous solution containing 0.8 kg of gentamicin and 0.8 kg of ampicillin, filter through a 0.22 μm filter membrane for sterilization, and feed into the fermenter.

[0063] 3) Samples were taken for microscopic examination at 94h, 95h, 96h, and 97h of the fermentation cycle, and 10, 13, 12, and 12 strains of Brevibacterium were observed under a 10x100x microscope. The number no longer increased, indicating that the growth of Brevibacterium was completely inhibited.

[0064] 4) The fermentation was terminated after 150 h, and the erythromycin A titer was detected to be 7558 u / ml.

[0065] Example 3

[0066] 1) During the erythromycin fermentation process of the example, microscopic examination revealed that microscopic bacilli were present at 70 h of the fermentation cycle, and 6 strains were observed under the field of view of a 10x100x microscope.

[0067] 2) Prepare an aqueous solution containing 1.1 kg of gentamicin and 1.1 kg of ampicillin, filter through a 0.22 μm filter membrane for sterilization, and feed into the fermenter.

[0068] 3) Samples were taken for microscopic examination at 72h, 73h, 74h, and 75h of the fermentation cycle, and 22, 24, 22, and 26 strains of Bacillus subtilis were observed under the field of view of 10x100 magnification microscope. The number no longer increased, indicating that the growth of Bacillus subtilis was completely inhibited.

[0069] 4) The fermentation was terminated after 150 h, and the erythromycin A titer was detected to be 6958 u / ml.

Claims

1. A fermentation method for continuously producing erythromycin using a contaminated fermentation broth, characterized in that The resistant Saccharopolyspora erythrae is used as the production bacterium, and antibiotics corresponding to the resistance are added to the bacterial fermentation liquid, wherein: The production bacteria is Saccharopolyspora erythraea with gentamicin resistance or ampicillin resistance; The antibiotic is gentamicin or ampicillin; The bacterial contaminant is selected from at least one of long bacillus and short bacillus.

2. According to the fermentation method according to claim 1, the resistant Saccharopolyspora erythraea is prepared by a corresponding antibiotic resistance screening method.

3. The fermentation method according to claim 1, characterized in that The gentamicin concentration for inhibiting bacterial infection is 3 μg / ml-12 μg / ml.

4. The fermentation method according to claim 1, characterized in that The ampicillin inhibits bacterial infection at a concentration of 3 μg / ml-12 μg / ml.

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