Fermentation medium, fermentation method and application of polymyxin B sulfate

Abstract

The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation medium, a fermentation method and application of polymyxin B sulfate. According to the method, carbon and nitrogen sources for fermentation are optimized, special amino acid is added, fed-batch components are optimized, the fermentation process is controlled, the adjusted fermentation culture medium and fermentation method meet the standards of EP8.0 and CP2015, the fermentation titer is high, the ratio of a fermentation product B1 / B2 is high, the purity of a target product is high, loss caused by follow-up refining or purification is reduced, and industrial popularization is facilitated.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation medium for polymyxin B sulfate, a fermentation method and its application. Background Art

[0002] Polymyxin B sulfate belongs to extracellular antibiotics, secreted in the fermentation broth, composed of a cyclic decapeptide and a fatty acyl tail, containing four components B1, B2, B3 and B1-I, having a significant curative effect on Gram-negative bacteria, and being able to neutralize the endotoxin produced by Gram-negative bacteria, reducing the pyrogenic reaction. It is considered to be the last line of defense for treating multi-drug resistant Gram-negative bacteria. Its antibacterial spectrum and clinical application are similar to those of polymyxin E, and it has an inhibitory or bactericidal effect on Gram-negative bacilli such as Escherichia coli, Pseudomonas aeruginosa, Paracolobactrum, Klebsiella pneumoniae, Acidophilus bacillus, Bordetella pertussis and Shigella dysenteriae, etc. Clinically, it is mainly used for infections caused by sensitive bacteria and urinary tract infections, meningitis, sepsis, burn infections and skin and mucous membrane infections caused by Pseudomonas aeruginosa, etc.

[0003] The chemical structure of polymyxin B is as follows:

[0004]

[0005] In China, the production of polymyxin B by fermentation started in the 1990s, with a relatively late start. The main optimization directions focused on strain screening and strain optimization. At present, the polymyxin B-producing bacterium Bacillus polymyxa has a fast metabolic rate, a short culture period, is sensitive to environmental conditions and process conditions, and it is difficult to precisely control the fermentation process. Moreover, the amount of flour added is large, the gelatinization time is long, and there will be incomplete gelatinization, which affects the quality of the nitrogen source in the fermentation broth. The main production method of polymyxin B sulfate is batch fermentation, with excessive carbon and nitrogen sources in the early stage, strong vegetative growth of the bacteria, insufficient in the later stage, and insufficient accumulation of polymyxin B. From the structure of polymyxin B, it contains multiple components such as B1, B2, B3, B1-I, etc., among which B1 is the main component. A high B1 / B2 ratio means a higher yield of the active ingredient in the production process, a higher purity of the target product, and a higher titer, which is beneficial to reducing the losses caused by subsequent refining or purification. However, the B1 / B2 ratio produced by the existing fermentation methods of polymyxin B sulfate is generally low. Based on the problems existing in the prior art, it is necessary to develop a new fermentation medium formula and fermentation process. Summary of the Invention

[0006] The purpose of the present invention is to provide a fermentation medium for polymyxin B sulfate, a fermentation method and its application, so as to solve the defects of low titer of polymyxin B sulfate, low content of active ingredients and low purity of the target product produced by the existing fermentation medium or fermentation method.

[0007] Polymyxin B sulfate is a multi-component substance, and B1, B2, B3 and B1-1 are its main components.

[0008] In a first aspect, in order to achieve the above object, the present invention provides a fermentation medium formulation, which is divided into a basic formulation composed of a fermentation carbon source, a nitrogen source and inorganic salts and a feeding formulation composed of a fermentation carbon-nitrogen source. The basic fermentation medium contains 5-15 g / L of soy protein concentrate, 25-50 g / L of maltodextrin, 3-15 g / L of yeast powder, 0.15-0.75 g / L of dipotassium hydrogen phosphate, 1.5-8 g / L of ammonium sulfate, 1-10 g / L of calcium carbonate, 5-12 g / L of antifoaming agent, 6-15 g / L of soybean oil, 0.1-2 g / L of L-leucine, 0.1-2 g / L of L-isoleucine, 0.2-1 g / L of lysine, 0.25-0.85 g / L of valine, 0.01-1 g / L of serine, and 0.2-1.6 g / L of L-threonine.

[0009] In another embodiment, the basic fermentation medium contains 8-12 g / L of soy protein concentrate, 30-45 g / L of maltodextrin, 4-11 g / L of yeast powder, 0.25-0.65 g / L of dipotassium hydrogen phosphate, 3.5-6 g / L of ammonium sulfate, 2-8 g / L of calcium carbonate, 6-10 g / L of antifoaming agent, 8-12 g / L of soybean oil, 0.2-1.5 g / L of L-leucine, 0.15-1.23 g / L of L-isoleucine, 0.35-0.78 g / L of lysine, 0.35-0.77 g / L of valine, 0.02-0.85 g / L of serine, and 0.5-1.2 g / L of L-threonine.

[0010] In a more preferred embodiment, the present invention also includes a feeding process, and the feeding medium contains 6-15 g / L of soy protein concentrate and 30-45 g / L of maltodextrin; preferably, the feeding medium contains 6-15 g / L of soy protein concentrate, 30-45 g / L of maltodextrin, 10-25 g / L of medium-temperature soybean cake powder, 10-17 g / L of corn steep liquor dry powder, 10-30 g / L of glucose, and 20-35 g / L of sucrose.

[0011] In a more preferred embodiment, the fermentation medium provided by the present invention contains 9.5 g / L of soy protein concentrate, 38 g / L of maltodextrin, 7 g / L of yeast powder, 0.36 g / L of dipotassium hydrogen phosphate, 4.5 g / L of ammonium sulfate, 5 g / L of calcium carbonate, 8 g / L of antifoaming agent, 10 g / L of soybean oil, 0.95 g / L of L-leucine, 0.15 - 1.23 g / L of L-isoleucine, 0.35 - 0.78 g / L of lysine, 0.35 - 0.77 g / L of valine, 0.02 - 0.85 g / L of serine, and 0.83 g / L of L-threonine; the feeding medium is 9.5 g / L of soy protein concentrate, 30 g / L of maltodextrin, 16 g / L of medium-temperature soybean cake powder, 13.5 g / L of corn steep liquor dry powder, 19 g / L of glucose, and 28 g / L of sucrose.

[0012] The basal medium contains: 15 - 30 g / L of low-temperature soybean flour, 15 - 30 g / L of glucose, 5 - 15 g / L of soy protein concentrate, 25 - 45 g / L of maltodextrin, 2 - 8 g / L of yeast powder, 0.05 - 0.6 g / L of dipotassium hydrogen phosphate, 3 - 6 g / L of ammonium sulfate, 1 - 6 g / L of calcium carbonate, 6 - 20 g / L of salad oil (soybean oil), 0.2 - 1.5 g / L of L-leucine, 0.15 - 1.23 g / L of L-isoleucine, 0.35 - 0.78 g / L of lysine, 0.35 - 0.77 g / L of valine, 0.02 - 0.85 g / L of serine, 0.02 - 0.1 g / L of L-threonine, 5 - 18 g / L of antifoaming agent, and the rest is water. The feeding formula is per liter of medium composition, 6 - 15 g / L of soy protein concentrate, 20 - 40 g / L of maltodextrin, and the rest is water.

[0013] In another embodiment, the basal formula contains: 15 - 30 g / L of low-temperature soybean flour, 8 - 12 g / L of soy protein concentrate, 30 - 45 g / L of maltodextrin, 15 - 30 g / L of glucose, 3 - 5 g / L of yeast powder, 0.1 - 0.3 g / L of dipotassium hydrogen phosphate, 3 - 6 g / L of ammonium sulfate, 3 - 6 g / L of calcium carbonate, 8 - 12 g / L of salad oil (soybean oil), 0.2 - 1.5 g / L of L-leucine, 0.15 - 1.23 g / L of L-isoleucine, 0.35 - 0.78 g / L of lysine, 0.35 - 0.77 g / L of valine, 0.02 - 0.85 g / L of serine, 0.02 - 0.1 g / L of L-threonine, and 6 - 10 g / L of antifoaming agent. The feeding formula is per liter of medium composition: 6 - 15 g / L of soy protein concentrate, 30 - 45 g / L of maltodextrin.

[0014] The solvent of the medium of the present invention is water, preferably deionized water or distilled water.

[0015] In the second aspect, the present invention provides a fermentation method for polymyxin B sulfate. The fermentation method is as follows.

[0016] 1) Inoculate the seed liquid of polymyxin B sulfate into the aforementioned fermentation medium, and conduct fermentation culture at 25 - 35°C, controlling the dissolved oxygen to be not less than 20%, the rotation speed to be 150 - 250 rpm, and the aeration ratio to be 1:0.6 - 1.2 VVM;

[0017] 2) Starting from the middle stage of fermentation, when the pH rises to 6 - 8.5, supplement the feeding solution composed of carbon and nitrogen sources, and simultaneously add ammonia water to control the fermentation pH at 5 - 7.5;

[0018] 3) Starting from 24 h of fermentation, sample every 6 h. When the fermentation time is more than 96 h and the detected titer starts to decrease, stop feeding. Sample and empty the fermenter when the pH rebounds to about 7.

[0019] Preferably, in the step 1), the inoculation amount of the strain is 8% - 12%.

[0020] Preferably, in the step 1), the initial rotation speed is 70 rpm, which is reduced to 62 - 65 rpm after feeding. When the pH during fermentation decreases to 6.35, control the pH at 6.35 ± 0.02.

[0021] Preferably, in the step 1), control the dissolved oxygen DO between 10% - 65%, and control the aeration ratio to be 1:0.75 - 1:1 VVM. When the aeration ratio is 1:0.75 VVM, the dissolved oxygen DO is between 35% - 45%.

[0022] Preferably, in the step 1), control the dissolved oxygen DO between 35% - 45%, and the aeration ratio is 1:0.75 VVM.

[0023] The culture medium of the basic formula adopts the sterilization method of single sterilization in the fermenter. Dissolve the basic fermentation medium in the fermenter and sterilize it at 115 - 125°C with pressure maintained for 25 - 35 min.

[0024] The culture medium of the feeding formula adopts the sterilization method of single sterilization in the feeding bottle. Dissolve the feeding medium composed of fermentation carbon and nitrogen sources in the feeding bottle and sterilize it at 115 - 125°C with pressure maintained for 15 - 20 min.

[0025] The feeding method of the fermentation process steps of the present invention adopts the method of variable flow addition, and adjusts the feeding speed according to the pH performance.

[0026] The fermentation cycle of the fermentation process steps of the present invention selects the time to empty the fermenter according to the pH, the bacterial metabolism situation and the detection result of the fermentation broth titer.

[0027] The strain producing polymyxin B used in the present invention is Paenibacillus polymyxa well-known to those skilled in the art, and the strain number is CGMCC 1.7667.

[0028] Thirdly, the present invention provides the application of the culture medium for fermenting polymyxin B sulfate described in the first aspect or the fermentation method described in the second aspect in the production of polymyxin B sulfate.

[0029] Compared with the prior art, the technical solution of the present invention optimizes the components of the culture medium, selects suitable carbon and nitrogen sources and amino acids, improves the utilization rate of bacteria, increases the B1 / B2 ratio, reduces the loss in the refining process, and improves the yield. Through the feeding process and a specific feeding culture medium, the yield of polymyxin B is further increased, the production cost is effectively reduced, and it is easy to be promoted in the industry. Detailed Embodiments

[0030] The following further elaborates the present invention in conjunction with embodiments, but the protection scope of the present invention cannot be considered limited to the following specific embodiments. Any process improvements made by those skilled in the art based on this method fall within the protection scope of the present invention.

[0031] The fermenters used in Examples 1 - 7 were Shanghai Guoqiang 50L fermenters, the seed tanks used were Shanghai Guoqiang 15L fermenters, and all other equipment used were conventional instruments and equipment. All the fermentation raw materials used were industrial grade / food grade raw materials, and the sources were purchased commercially.

[0032] The formula of the seed culture medium used in the following examples is

[0033] Composition per liter of the culture medium: 15 g of low-temperature soybean powder, 15 g of glucose, 2 g of yeast powder, 0.05 g of dipotassium hydrogen phosphate, 3 g of ammonium sulfate, 6 g of salad oil, 5 g of antifoaming agent, and the rest is water.

[0034] The culture conditions for the seed tanks used were: temperature 29 - 32°C, stirring 100 - 250 rpm, aeration ratio 1:0.8 - 1.0 VVM, and fermentation cycle 10 - 24 h.

[0035] The detection instrument for the polymyxin B sulfate fermentation broth of the present invention: liquid chromatograph, product number Agilent 1260

[0036] The detection chromatographic conditions are as follows:

[0037] Chromatographic column: Chrom Core AQ C18, 250 mm × 4.6 mm

[0038] Mobile phase A: sodium thiosulfate solution - acetonitrile - water (70:21:9)

[0039] Mobile phase B: acetonitrile - water (7:3)

[0040] Flow rate: 1.0 ml / min

[0041] Column temperature: 25°C

[0042] Detection wavelength: 215 nm

[0043] Injection volume: 20 μl

[0044] The elution procedure is as follows:

[0045] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 60 100 0 61 10 90 65 10 90 65.1 100 0 75 100 0

[0046] Example 1

[0047] Prepare the fermentation medium according to the following formula. The medium ratio: The basic formula is that each liter of the medium consists of: 80 g of wheat flour, 0.04 g of thermotolerant α-amylase, 4 g of yeast powder, 0.15 g of dipotassium hydrogen phosphate, 4.5 g of ammonium sulfate, 3 g of calcium carbonate, 10 g of salad oil, 8 g of antifoaming agent, 0.1 g of L-leucine, 0.075 g of L-isoleucine, 0.075 g of L-threonine, and the rest is water.

[0048] The fermentation process is carried out according to the following specific steps:

[0049] Dissolve the above materials in an appropriate amount of water, add the antifoaming agent, adjust the pH to 6.95 - 7 with pre-sterilization lye, then add calcium carbonate, sterilize at 121 °C and keep the pressure for 30 min, and measure the post-sterilization pH to be 6.8 - 7.2.

[0050] Culture the seed liquid producing polymyxin B according to the above seed process, inoculate it into the above fermentation medium at an inoculation amount of 8% - 12%, set the rotation speed to 100 rpm, the aeration ratio to 1:1 VVM, the dissolved oxygen DO ≥ 25%, the fermentation temperature to 29 ± 1 °C, the tank pressure to 0.05 MPa, the flow rate to 102 m 3 / h, and the fermentation period is 48 - 66 h. At the start of fermentation, when the pH rebounds to 7.5, the fermentation period is based on microscopic examination of the aging of the bacteria, the pH rises rapidly, and then the fermentation broth is discharged. The titer of the fermentation broth is detected to reach 6420 u / mL, and at the same time, B1 / B2 = 3:1.

[0051] Example 2

[0052] Prepare the fermentation medium according to the following formula. The medium ratio: The basic formula is that each liter of the medium consists of: 80 g of wheat flour, 0.04 g of thermotolerant α-amylase, 4 g of yeast powder, 0.15 g of dipotassium hydrogen phosphate, 4.5 g of ammonium sulfate, 3 g of calcium carbonate, 10 g of salad oil, 8 g of antifoaming agent, 0.2 g of L-leucine, 0.15 g of L-isoleucine, 0.075 g of L-threonine, 0.35 g of lysine, 0.35 g of valine, 0.02 g of serine, and the rest is water.

[0053] The fermentation process is carried out according to the following specific steps:

[0054] Dissolve the above materials in an appropriate amount of water, add an antifoaming agent, adjust the pH to 6.95 - 7 with pre-sterilization lye, then add calcium carbonate, sterilize at 121°C under pressure for 30 min, and measure the pH after sterilization to be 6.8 - 7.2.

[0055] Cultivate the seed liquid producing polymyxin B according to the above seed process, inoculate it into the above fermentation medium at an inoculation amount of 8% - 12%, set the rotation speed to 100 rpm, the aeration ratio to 1:1 VVM, the dissolved oxygen DO ≥ 25%, the fermentation temperature to 29 ± 1°C, the tank pressure to 0.05 MPa, and the flow rate to 102 m 3 / h, and the fermentation period is 48 - 66 h. At the start of fermentation, when the pH rebounds to 7.5, the fermentation period is determined according to the aging of the bacteria observed under the microscope, and when the pH rises rapidly, the fermentation broth is discharged. At the end of fermentation, the titer of the fermentation broth is detected to reach 8830 u / mL, and at the same time, B1 / B2 = 2.2:1.

[0056] Compared with Example 1, in Example 2, the dosages of L-leucine, L-isoleucine, and L-threonine were adjusted, and at the same time, lysine, valine, and serine were added to the fermentation medium, resulting in an increase in the titer of the fermentation broth and a decrease in the ratio of B1 / B2.

[0057] Example 3

[0058] Prepare the fermentation medium according to the following formula. The medium ratio: The basic formula is that each liter of the medium consists of: 80 g of wheat flour, 0.04 g of thermotolerant α-amylase, 4 g of yeast powder, 0.15 g of dipotassium hydrogen phosphate, 4.5 g of ammonium sulfate, 3 g of calcium carbonate, 10 g of salad oil, 8 g of antifoaming agent, 1.5 g of L-leucine, 1.23 g of L-isoleucine, 0.075 g of L-threonine, 0.78 g of lysine, 0.77 g of valine, 0.85 g of serine, and the rest is water.

[0059] The fermentation process is carried out according to the following specific steps:

[0060] Dissolve the above materials in an appropriate amount of water, add an antifoaming agent, adjust the pH to 6.95 - 7 with pre-sterilization lye, then add calcium carbonate, sterilize at 121°C under pressure for 30 min, and measure the pH after sterilization to be 6.8 - 7.2.

[0061] Cultivate the seed liquid producing polymyxin B according to the above seed process, inoculate it into the above fermentation medium at an inoculation amount of 8% - 12%, set the rotation speed to 100 rpm, the aeration ratio to 1:1 VVM, the dissolved oxygen DO ≥ 25%, the fermentation temperature to 29 ± 1°C, the tank pressure to 0.05 MPa, and the flow rate to 102 m 3 / h, and the fermentation period is 48 - 66 h. At the start of fermentation, when the pH rebounds to 7.5, the fermentation period is determined according to the aging of the bacteria observed under the microscope, and when the pH rises rapidly, the fermentation broth is discharged. The titer of the fermentation broth is detected to reach 10120 u / mL, and at the same time, B1 / B2 = 8.5:1.

[0062] Compared with Example 2, in Example 3, the dosages of L-leucine, L-isoleucine, L-threonine, lysine, valine, and serine were adjusted, resulting in a simultaneous increase in the titer of the fermentation broth and the ratio of B1 / B2.

[0063] Example 4

[0064] Prepare the fermentation medium according to the following formula. The medium ratio: The basic formula is per liter of medium composition: soy protein concentrate (65%) 9.5 g, maltodextrin 38 g, low-temperature soybean flour 15 - 30 g, glucose 15 - 30 g, wheat flour 80 g, thermostable α-amylase 0.04 g, yeast powder 4 g, dipotassium hydrogen phosphate 0.15 g, ammonium sulfate 4.5 g, calcium carbonate 3 g, salad oil 10 g, antifoaming agent 8 g, L-leucine 1.5 g, L-isoleucine 1.23 g, L-threonine 0.075 g, lysine 0.78 g, valine 0.77 g, serine 0.85 g, and the rest is water.

[0065] The fermentation process is implemented according to the following specific steps:

[0066] Dissolve the above materials in an appropriate amount of water, add the antifoaming agent, adjust the pH to 6.95 - 7 with pre-sterilization lye, then add calcium carbonate, sterilize at 121°C and maintain the pressure for 30 min, and measure the post-sterilization pH to be 6.8 - 7.2.

[0067] Culture the seed liquid producing polymyxin B according to the above seed process, inoculate it into the above fermentation medium at an inoculation amount of 8% - 12%, set the rotation speed to 100 rpm, the aeration ratio to 1:1 VVM, the dissolved oxygen DO ≥ 25%, the fermentation temperature to 29 ± 1°C, the tank pressure to 0.05 MPa, the flow rate to 102 m 3 / h, and the fermentation period is 48 - 66 h. At the start of fermentation, when the pH rebounds to 7.5, the fermentation period is based on microscopic examination of the aging of the bacterial cells, the pH rises rapidly, and then the fermentation broth is discharged. The titer of the fermentation broth is detected to reach 13350 u / mL, and at the same time, B1 / B2 = 6.2:1.

[0068] Compared with Example 3, in Example 4, in terms of carbon and nitrogen sources in the fermentation medium, soy protein concentrate, maltodextrin, low-temperature soybean flour, and glucose were increased. The titer of the fermentation broth in Example 4 increased compared with that in Example 3, but the ratio of B1 / B2 decreased.

[0069] Example 5

[0070] Prepare the fermentation medium according to the following formula, using the optimized medium ratio obtained in the present invention: The basic formula is the composition per liter of the medium: soy protein concentrate (65%) 9.5 g, maltodextrin 38 g, low-temperature soy flour 15 - 30 g, glucose 15 - 30 g, wheat flour 80 g, heat-resistant α-amylase 0.04 g, yeast powder 4 g, dipotassium hydrogen phosphate 0.15 g, ammonium sulfate 4.5 g, calcium carbonate 3 g, salad oil 10 g, antifoaming agent 8 g, L-leucine 1.5 g, L-isoleucine 1.23 g, L-threonine 0.075 g, lysine 0.78 g, valine 0.77 g, serine 0.85 g, and the rest is water. The feeding formula is the composition per liter of the medium: soy protein concentrate 9.5 g, maltodextrin 38 g, and the rest is water.

[0071] The fermentation process is carried out according to the following specific steps:

[0072] Dissolve the above materials in an appropriate amount of water, add the antifoaming agent, adjust the pH to 6.95 - 7 with pre-sterilization lye, then add calcium carbonate, sterilize at 121 °C and maintain the pressure for 30 min, and measure the pH after sterilization to be 6.8 - 7.2.

[0073] Cultivate the seed liquid producing polymyxin B according to the above seed process, inoculate it into the above fermentation medium at an inoculation amount of 8% - 12%, set the rotation speed to 100 rpm, the aeration ratio to 1:1 VVM, the dissolved oxygen DO ≥ 25%, the fermentation temperature to 29 ± 1 °C, the tank pressure to 0.05 MPa, the flow rate to 102 m3 / h, and the fermentation period to 48 - 66 h. At the start of fermentation, start feeding according to the pH rebound of 0.01 - 0.5, and when the pH rebounds to 7.5, end the fermentation according to the aging of the bacteria observed under the microscope and the rapid rise of the pH. Detect that the titer of the fermentation broth reaches 15500 u / mL, and at the same time B1 / B2 = 7.3:1.

[0074] Compared with Example 4, in Example 5, 9.5 g of soy protein concentrate and 38 g of maltodextrin are added to the feeding solution, and the titer of the fermentation broth is further increased, and the ratio of B1 / B2 is further increased.

[0075] Example 6

[0076] Prepare the fermentation medium according to the following formula, using the optimized medium ratio obtained in the present invention: The basic formula is the composition per liter of the medium: soy protein concentrate (65%) 9.5 g, maltodextrin 38 g, low-temperature soy flour 15 - 30 g, glucose 15 - 30 g, yeast powder 4 g, dipotassium hydrogen phosphate 0.15 g, ammonium sulfate 4.5 g, calcium carbonate 5 g, salad oil 10 g, antifoaming agent 8 g, L-leucine 1.5 g, L-isoleucine 1.23 g, L-threonine 0.075 g, lysine 0.78 g, valine 0.77 g, serine 0.85 g, and the rest is water. The feeding formula is the composition per liter of the medium: soy protein concentrate 9.5 g, maltodextrin 38 g, and the rest is water.

[0077] The fermentation process is carried out according to the following specific steps:

[0078] Dissolve the above materials in an appropriate amount of water, add an antifoaming agent, adjust the pH to 6.95 - 7 with pre-sterilization lye, then add calcium carbonate, sterilize at 121°C and maintain pressure for 30 min, and the volume after sterilization is 27 L. The pH after sterilization is measured to be 6.8 - 7.2.

[0079] Cultivate the seed liquid producing polymyxin B according to the above seed process, inoculate it into the above fermentation medium at an inoculation amount of 8% - 12%, set the rotation speed to 70 rpm, and reduce it to 62 - 65 rpm after feeding. The aeration ratio is 1:0.75 VVM, the dissolved oxygen DO is between 35% and 45%, the fermentation temperature is 29 ± 1°C, the tank pressure is 0.05 MPa, and the flow rate is 102 m 3 / h, and the fermentation period is 48 - 66 h. At the start of fermentation, samples are taken every 6 h starting from 24 h. Feed is added according to the pH rebound of 0.01 - 0.5. When the fermentation time is more than 96 h and the detected titer starts to decrease, stop feeding. Samples are taken and the tank is emptied when the pH rises to about 7. At the end of fermentation, the titer of the fermentation broth is detected to reach 16358 u / mL, and at the same time, B1 / B2 = 9.2:1.

[0080] Compared with Example 5, in Example 6, the fermentation medium reduces wheat flour and heat-resistant α-amylase, increases the dosage of calcium carbonate, adjusts the filling volume, dissolved oxygen, and flow rate of the fermentation process, and the ratio of product B1 / B2 and the titer of fermentation are improved simultaneously.

[0081] Example 7

[0082] Prepare the fermentation medium according to the following formula, using the optimized medium ratio of the present invention: The basic formula is the composition per liter of the medium: soy protein concentrate (65%) 9.5 g, maltodextrin 38 g, yeast powder 7 g, dipotassium hydrogen phosphate 0.36 g, ammonium sulfate 4.5 g, calcium carbonate 5 g, salad oil 10 g, antifoaming agent 8 g, L-leucine 0.95 g, L-isoleucine 1.23 g, L-threonine 0.83 g, lysine 0.78 g, valine 0.77 g, serine 0.85 g, and the rest is water. The feeding formula is the composition per liter of the medium: soy protein concentrate 9.5 g, maltodextrin 30, medium-temperature soybean cake powder 16 g, corn steep liquor dry powder 13.5 g, glucose 19 g, sucrose 28 g, and the rest is water.

[0083] The fermentation process is carried out according to the following specific steps:

[0084] Dissolve the above materials in an appropriate amount of water, add an antifoaming agent, adjust the pH to 6.95 - 7 with pre-sterilization lye, then add calcium carbonate, sterilize at 121°C and maintain pressure for 30 min, and the volume after sterilization is 27 L. The pH after sterilization is measured to be 6.8 - 7.2.

[0085] Cultivate the seed liquid for producing polymyxin B according to the above seed process, inoculate it into the above fermentation medium at an inoculation amount of 8%-12%, set the rotation speed to 70 rpm, and reduce it to 62-65 rpm after feeding. The aeration ratio is 1:0.75 VVM, the dissolved oxygen DO is between 35%-65%, the fermentation temperature is 29±1°C, the tank pressure is 0.05 MPa, and the flow rate is 102 m 3 / h, and the fermentation cycle is 48-66 h. At the beginning of fermentation, samples are taken every 6 h starting from 24 h. Feeding starts according to the pH rebound of 0.01-0.5. When the fermentation time is more than 96 h and the detected titer starts to decrease, feeding stops. Sampling and discharging the tank are carried out when the pH rebounds to about 7. After fermentation ends, the titer of the fermentation broth is detected to reach 18132 u / mL, and at the same time B1 / B2 = 17.0:1.

[0086] Compared with Example 6, in Example 7, the dosage of L-threonine was adjusted, the feeding medium was further optimized, the medium-temperature soybean cake powder, corn steep liquor dry powder, glucose and sucrose were increased, and the titer of fermentation was further improved. The ratio of B1 / B2 in the fermentation product was as high as 17.0:1.

[0087] Finally, it should be noted that the above are only the embodiments of the present application, which are only used to illustrate the technical solutions of the present invention and are not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A culture medium for fermenting polymyxin B sulfate, characterized in that, Containing 5 - 15 g / L of soy protein concentrate, 25 - 50 g / L of maltodextrin, 3 - 15 g / L of yeast powder, 0.15 - 0.75 g / L of dipotassium hydrogen phosphate, 1.5 - 8 g / L of ammonium sulfate, 1 - 10 g / L of calcium carbonate, 5 - 12 g / L of antifoaming agent, 6 - 15 g / L of soybean oil, 0.1 - 2 g / L of L - leucine, 0.1 - 2 g / L of L - isoleucine, 0.2 - 1 g / L of lysine, 0.25 - 0.85 g / L of valine acid, 0.01 - 1 g / L of serine, 0.2 - 1.6 g / L of L - threonine.

2. The culture medium according to claim 1, characterized in that, Containing 8 - 12 g / L of soy protein concentrate, 30 - 45 g / L of maltodextrin, 4 - 11 g / L of yeast powder, 0.25 - 0.65 g / L of dipotassium hydrogen phosphate, 3.5 - 6 g / L of ammonium sulfate, 2 - 8 g / L of calcium carbonate, 6 - 10 g / L of antifoaming agent, 8 - 12 g / L of soybean oil, 0.2 - 1.5 g / L of L - leucine, 0.15 - 1.23 g / L of L - isoleucine, 0.35 - 0.78 g / L of lysine, 0.35 - 0.77 g / L of valine acid, 0.02 - 0.85 g / L of serine, 0.5 - 1.2 g / L of L - threonine.

3. A culture medium for fermenting polymyxin B sulfate, characterized in that, Containing 15 - 30 g / L of low - temperature soybean flour, 15 - 30 g / L of glucose, 5 - 15 g / L of soy protein concentrate, 25 - 45 g / L of maltodextrin, 2 - 8 g / L of yeast powder, 0.05 - 0.6 g / L of dipotassium hydrogen phosphate, 3 - 6 g / L of ammonium sulfate, 1 - 6 g / L of calcium carbonate, 6 - 20 g / L of salad oil (soybean oil), 0.2 - 1.5 g / L of L - leucine, 0.15 - 1.23 g / L of L - isoleucine, 0.35 - 0.78 g / L of lysine, 0.35 - 0.77 g / L of valine, 0.02 - 0.85 g / L of serine, 0.02 - 0.1 g / L of L - threonine, 5 - 18 g / L of antifoaming agent; Preferably, the culture medium components include 15 - 30 g / L of low - temperature soybean flour, 8 - 12 g / L of soy protein concentrate, 30 - 45 g / L of maltodextrin, 15 - 30 g / L of glucose, 3 - 5 g / L of yeast powder, 0.10 - 0.3 g / L of dipotassium hydrogen phosphate, 3 - 6 g / L of ammonium sulfate, 3 - 6 g / L of calcium carbonate, 8 - 12 g / L of salad oil (soybean oil), 0.2 - 1.5 g / L of L - leucine, 0.15 - 1.23 g / L of L - isoleucine, 0.35 - 0.78 g / L of lysine, 0.35 - 0.77 g / L of valine, 0.02 - 0.85 g / L of serine, 0.02 - 0.1 g / L of L - threonine, 6 - 10 g / L of antifoaming agent.

4. The culture medium according to claims 1 - 3, characterized in that, The culture medium also includes a feeding process, and the feeding medium contains 6 - 15 g / L of soy protein concentrate and 30 - 45 g / L of maltodextrin; preferably, the feeding medium contains 6 - 15 g / L of soy protein concentrate, 30 - 45 g / L of maltodextrin, 10 - 25 g / L of medium - temperature soybean cake powder, 10 - 17 g / L of dry corn steep liquor, 10 - 30 g / L of glucose, and 20 - 35 g / L of sucrose.

5. The culture medium according to any one of claims 1, 2 or 4, characterized in that It contains 9.5 g / L of soy protein concentrate, 38 g / L of maltodextrin, 7 g / L of yeast powder, 0.36 g / L of dipotassium hydrogen phosphate, 4.5 g / L of ammonium sulfate, 5 g / L of calcium carbonate, 8 g / L of antifoaming agent, 10 g / L of soybean oil, 0.95 g / L of L-leucine, 0.15 - 1.23 g / L of L-isoleucine, 0.35 - 0.78 g / L of lysine, 0.35 - 0.77 g / L of valine, 0.02 - 0.85 g / L of serine, and 0.83 g / L of L-threonine; the feeding medium contains 9.5 g / L of soy protein concentrate, 30 g / L of maltodextrin, 16 g / L of medium-temperature soybean cake powder, 13.5 g / L of corn steep liquor dry powder, 19 g / L of glucose, and 28 g / L of sucrose.

6. A fermentation method of polymyxin B sulfate, characterized in that, The fermentation method is as follows 1) Inoculate the seed liquid of polymyxin B sulfate into the fermentation medium described in any one of claims 1 - 5, and carry out fermentation culture at 25 - 35 °C, control the dissolved oxygen not less than 20%, the rotation speed is 50 - 150 rpm, and the ventilation ratio is 1:0.6 - 1.2 VVM; 2) Starting from the middle stage of fermentation, when the pH rises to 6 - 8.5, supplement the feeding liquid composed of carbon and nitrogen sources, and at the same time supplement ammonia water to control the fermentation pH at 5 - 7.5; 3) Start sampling every 6 h after 24 h of fermentation, the fermentation time is more than 96 h, stop feeding when the detected titer begins to decrease, and sample and discharge the tank when the pH rebounds to about 7.

7. The fermentation method according to claim 6, characterized in that, The inoculation amount of the strain is 8% - 12%.

8. The fermentation method according to claim 5 or 6, characterized in that, The initial rotation speed is 70 rpm, and it is reduced to 62 - 65 rpm after feeding. When the pH during fermentation drops to 6.35, control the pH at 6.35 ± 0.

02.

9. The fermentation method according to any one of claims 5 or 6, characterized in that Control the dissolved oxygen DO between 10% - 65%, and control the ventilation ratio at 1:0.75 - 1:1 VVM.

10. The application of the medium for fermenting polymyxin B sulfate described in any one of claims 1 - 5 or the fermentation method described in any one of claims 6 - 9 in the production of polymyxin B sulfate.

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