A method for preparing a fermentation broth with increased content of polymyxin b effective components

Abstract

The application belongs to the technical field of microbial medicine, and particularly discloses a preparation method of a fermentation liquor with improved content of effective components of polymyxin B. The fermentation process is precisely controlled by adding carnosine as a key substance, adding glucose, designing a culture medium formula, and controlling temperature and pH in stages, so that the fermentation unit and the content of effective components are effectively improved, and thus the quality of the fermentation liquor is improved. Compared with a control example, the fermentation unit and the content of effective components are obviously improved, the fermentation unit is increased by 29.42% at most, and the effective components are increased by 12.32%.

Description

Technical Field

[0001] This invention relates to the field of microbial pharmaceutical technology, and in particular to a method for preparing fermentation broth that increases the content of the effective component of polymyxin B. Background Technology

[0002] Polymyxin B is a class of relatively large lipopeptide molecules composed of four main components: B1, B2, B3, and B1-I. Polymyxin B is a polypeptide antibiotic against Gram-negative bacilli. It inhibits the growth of Gram-negative bacteria by altering membrane structure, causing leakage of small molecules, and binding to the lipid portion of bacterial lipopolysaccharides, inducing pores in the epithelial cell membrane. It exhibits strong inhibitory and bactericidal effects against almost all Gram-negative bacteria, such as *Escherichia coli*, *Pseudomonas aeruginosa*, *Paracylpyridae*, *Klebsiella pneumoniae*, *Acidophilus*, *Bordetella pertussis*, and *Shigella*, with a particularly significant effect against *Pseudomonas aeruginosa*. Due to its excellent antibacterial activity against Gram-negative drug-resistant bacteria, polymyxin B has become a research hotspot in the field of anti-drug-resistant bacterial drugs in recent years.

[0003] Because polymyxin B is a multi-component product, controlling the content of each component is a challenge in the fermentation process. Specifically, the content of the effective components (total content of B1, B2, B3, and B1-I) must reach over 80%, with B3 ≤ 6%, B1-I ≤ 18%, maximum single impurity ≤ 3%, and total impurities ≤ 17%. The quality of the fermentation broth is a key factor affecting the final product. Meeting the requirements for the content of each component in the fermentation broth, and having a high sum of effective component content and high fermentation unit, makes extraction and purification easier and yields higher results, providing data support for cleaner production.

[0004] Currently, the fermentation level of polymyxin B in my country is relatively low, with an industry fermentation unit of approximately 2 g / L, while in 2007, the fermentation unit in foreign countries had already reached 2.8 g / L, indicating a significant gap. Furthermore, research on increasing the content of effective components and reducing impurities with polymyxin B is relatively limited, the factors affecting the total content of effective components are unclear, and the content in the fermentation broth is generally low. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies and provide a method for preparing fermentation broth that increases the content of polymyxin B effective components, thereby increasing the content of polymyxin B fermentation units and effective components in the fermentation broth.

[0006] To achieve the above objectives, the present invention is implemented according to the following technical solution:

[0007] A method for preparing fermentation broth with increased content of polymyxin B active components includes the following steps:

[0008] S1. Inoculate the well-grown Bacillus polymyxa seed culture into the fermentation medium at a volume of 10% of the fermentation tank volume after inoculation. Before inoculation, the aeration rate is 0.6 vvm, the tank pressure is 0.04 MPa-0.06 MPa, the rotation speed is 200-500 rpm, and the dissolved oxygen is 30 ≤ dissolved oxygen ≤ 40%.

[0009] S2. During fermentation from 0h to 12h, control the temperature inside the fermenter at 30-32℃, and add feed one to control the pH inside the fermenter at 6.5-6.7. During fermentation from 13h to 60h, control the temperature inside the fermenter at 27-29℃, and add feed two at 0.0001-0.0003 times the volume of fermentation liquid every hour, while adding feed one to control the pH inside the fermenter at 6.1-6.3.

[0010] Furthermore, it also includes:

[0011] S3. After fermentation for 30 hours, feed 0.002-0.004 times the fermentation volume of feed 3 at a constant rate every hour, and culture for 60 hours.

[0012] Further, the fermentation medium in step S1 is composed of the following components by weight per 100 mL: 6.0 g-8.0 g whole wheat flour, 0.01 g-0.02 g α-amylase, 0.3 g-0.5 g glucose, 1.5 g-2.5 g low-temperature soybean flour, 0.4 g-0.6 g corn steep liquor, 0.6 g-0.8 g ammonium sulfate, 0.025 g-0.035 g potassium dihydrogen phosphate, 0.05 g-0.15 g soybean lecithin, 0.3 g-0.5 g calcium carbonate, 0.1 g-0.2 g defoamer, and the remainder is water.

[0013] Furthermore, the feed material is ammonia water with a concentration of 20%.

[0014] Furthermore, the second feed is a sterilized carnosine solution with a concentration of 5% (W / V).

[0015] Furthermore, the supplemental material three is a sterilized 50% (w / v) glucose solution.

[0016] Preferably, the fermentation medium in step S1 is composed of the following components by weight per 100 mL: whole wheat flour 7.0, α-amylase 0.015, glucose 0.4, low-temperature soybean flour 2.0, corn steep liquor 0.5, ammonium sulfate 0.7, potassium dihydrogen phosphate 0.03, soybean lecithin 0.1, calcium carbonate 0.4, defoamer 0.15, and the remainder is water.

[0017] Preferably, during the fermentation period of 0h-12h, the temperature inside the fermentation tank is controlled at 31℃; during the fermentation period of 13h-60h, the temperature inside the fermentation tank is controlled at 28℃.

[0018] Preferably, the pH in the fermenter is controlled at 6.6 during the fermentation period of 0h-12h; and the pH in the fermenter is controlled at 6.2 during the fermentation period of 13h-60h.

[0019] Compared with existing technologies, this invention improves the quality of fermentation broth by precisely controlling the fermentation process through the addition of key substances such as carnosine and glucose, designing the culture medium formula, and controlling the temperature and pH in stages. This precise control effectively increases the fermentation unit and the content of effective components. Compared with the control example, both the fermentation unit and the content of effective components are significantly increased, with the fermentation unit increasing by up to 29.42% and the effective component increasing by 12.32%. Attached Figure Description

[0020] Figure 1 This is the high-performance liquid chromatogram of the fermentation broth from Example 4;

[0021] Figure 2 This is a high-performance liquid chromatogram of the fermentation broth from control example 1. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0023] Example 1

[0024] Step (1)

[0025] Preparation of fermentation medium (in g / 100mL): whole wheat flour 7.0, α-amylase 0.015, glucose 0.4, low-temperature soybean flour 2.0, corn steep liquor 0.5, ammonium sulfate 0.7, potassium dihydrogen phosphate 0.03, soybean lecithin 0.1, calcium carbonate 0.4, defoamer 0.15, the remainder being water;

[0026] Step (2)

[0027] The well-grown Bacillus polymyxa seed culture was inoculated into the above fermentation medium at a volume of 10% of the fermentation tank volume after inoculation. Before inoculation, the following conditions were controlled in the fermentation tank: aeration ratio of 0.6 vvm, tank pressure of 0.04 MPa-0.06 MPa, and rotation speed of 200-500 rpm. The dissolved oxygen was controlled at 30%-40% by adjusting the rotation speed.

[0028] Step (3)

[0029] Fermentation cycle 0h-12h: temperature controlled at 30℃, pH controlled at 6.7 by adding 20% ​​ammonia. Fermentation cycle 13h-60h: temperature controlled at 29℃, adding sterilized 5% (w / v) carnosine solution (0.0001 times the fermentation volume per hour), and simultaneously controlling the pH at 6.3 by adding 20% ​​ammonia. After 60h of cultivation, the final fermentation broth was obtained. The final fermentation broth was analyzed by high-performance liquid chromatography (HPLC), and the results are shown in the table below.

[0030]

[0031] Example 2

[0032] Step (1)

[0033] Preparation of fermentation medium (in g / 100mL): whole wheat flour 7.0, α-amylase 0.015, glucose 0.4, low-temperature soybean flour 2.0, corn steep liquor 0.5, ammonium sulfate 0.7, potassium dihydrogen phosphate 0.03, soybean lecithin 0.1, calcium carbonate 0.4, defoamer 0.15, the remainder being water;

[0034] Step (2)

[0035] The well-grown Bacillus polymyxa seed culture was inoculated into the above fermentation medium at a volume of 10% of the fermentation tank volume after inoculation. Before inoculation, the following conditions were controlled in the fermentation tank: aeration ratio of 0.6 vvm, tank pressure of 0.04 MPa-0.06 MPa, and rotation speed of 200-500 rpm. The dissolved oxygen was controlled at 30%-40% by adjusting the rotation speed.

[0036] Step (3)

[0037] Fermentation cycle 0h-12h: temperature controlled at 31℃, pH controlled at 6.6 by adding 20% ​​ammonia. Fermentation cycle 13h-60h: temperature controlled at 28℃, adding sterilized 5% (w / v) carnosine solution (0.0002 times the fermentation volume per hour), and simultaneously controlling the pH at 6.2 by adding 20% ​​ammonia. After 60h of cultivation, the final fermentation broth was obtained. The final fermentation broth was analyzed by high-performance liquid chromatography (HPLC), and the results are shown in the table below.

[0038]

[0039] Example 3

[0040] Step (1)

[0041] Preparation of fermentation medium (in g / 100mL): whole wheat flour 7.0, α-amylase 0.015, glucose 0.4, low-temperature soybean flour 2.0, corn steep liquor 0.5, ammonium sulfate 0.7, potassium dihydrogen phosphate 0.03, soybean lecithin 0.1, calcium carbonate 0.4, defoamer 0.15, the remainder being water;

[0042] Step (2)

[0043] The well-grown Bacillus polymyxa seed culture was inoculated into the above fermentation medium at a volume of 10% of the fermentation tank volume after inoculation. Before inoculation, the following conditions were controlled in the fermentation tank: aeration ratio of 0.6 vvm, tank pressure of 0.04 MPa-0.06 MPa, and rotation speed of 200-500 rpm. The dissolved oxygen was controlled at 30%-40% by adjusting the rotation speed.

[0044] Step (3)

[0045] During the fermentation cycle from 0h to 12h: the temperature was controlled at 31℃, and the pH was maintained at 6.6 by adding 20% ​​ammonia water. During the fermentation cycle from 13h to 60h: the temperature was controlled at 28℃, and a sterilized 5% (w / v) carnosine solution was added (0.0002 times the fermentation volume per hour), while the pH was maintained at 6.2 by adding 20% ​​ammonia water.

[0046] Step (4)

[0047] After a 30-hour fermentation cycle, a sterilized 50% (w / v) glucose solution was added at a constant rate (0.002 times the fermentation volume per hour). The mixture was cultured for 60 hours to obtain the final fermentation broth. The final fermentation broth was analyzed by high-performance liquid chromatography (HPLC), and the results are shown in the table below.

[0048]

[0049] Example 4

[0050] Step (1)

[0051] Preparation of fermentation medium (in g / 100mL): whole wheat flour 7.0, α-amylase 0.015, glucose 0.4, low-temperature soybean flour 2.0, corn steep liquor 0.5, ammonium sulfate 0.7, potassium dihydrogen phosphate 0.03, soybean lecithin 0.1, calcium carbonate 0.4, defoamer 0.15, the remainder being water;

[0052] Step (2)

[0053] The well-grown Bacillus polymyxa seed culture was inoculated into the above fermentation medium at a volume of 10% of the fermentation tank volume after inoculation. Before inoculation, the following conditions were controlled in the fermentation tank: aeration ratio of 0.6 vvm, tank pressure of 0.04 MPa-0.06 MPa, and rotation speed of 200-500 rpm. The dissolved oxygen was controlled at 30%-40% by adjusting the rotation speed.

[0054] Step (3)

[0055] During the fermentation cycle from 0h to 12h: the temperature was controlled at 31℃, and the pH was maintained at 6.6 by adding 20% ​​ammonia water. During the fermentation cycle from 13h to 60h: the temperature was controlled at 28℃, and a sterilized 5% (w / v) carnosine solution was added (0.0002 times the fermentation volume per hour), while the pH was maintained at 6.2 by adding 20% ​​ammonia water.

[0056] Step (4)

[0057] After a 30-hour fermentation cycle, a sterilized 50% (w / v) glucose solution was added at a constant rate (0.003 times the fermentation volume per hour). The mixture was cultured for 60 hours to obtain the final fermentation broth. The final fermentation broth was analyzed by high-performance liquid chromatography (HPLC), and the polymyxin B peak and yield were as follows: Figure 1 As shown in Table 1:

[0058] Table 1

[0059]

[0060] The principle of this invention is as follows: During the early stage of fermentation (0-12 hours), a relatively high temperature of 30-32℃ is controlled, which is conducive to the rapid growth of the initial bacterial count. During the rapid growth process, the pH gradually decreases, and ammonia water is added to control the pH at 6.5-6.7, which is suitable for rapid bacterial growth. From 13-60 hours, through fed-batch feeding and pH-controlled feeding, the pH is controlled to 6.1-6.3, which is suitable for the synthesis of polymyxin B, effectively increasing the content of effective components B1 and B2. The temperature is then lowered to 27-29℃, which can reduce the metabolic rate of the bacteria and reduce the excessive consumption of nutrients. This allows the bacteria to synthesize polymyxin B at a more suitable pH and temperature, which can significantly increase the polymyxin B unit. After 30 hours of fermentation, the growth rate of fermentation units begins to decline. Glucose is added at a constant rate to supplement and provide an appropriate amount of carbon source, which can effectively extend the high-unit fermentation cycle, and finally, the unit output and the content of effective components are significantly increased.

[0061] Furthermore, in order to verify the effect of the present invention on the quality of fermentation broth through the control of pH, temperature and feeding, the following control examples were set up.

[0062] Compare with Example 1

[0063] Step (1)

[0064] Preparation of fermentation medium (in g / 100mL): whole wheat flour 7.0, α-amylase 0.015, glucose 0.4, low-temperature soybean flour 2.0, corn steep liquor 0.5, ammonium sulfate 0.7, potassium dihydrogen phosphate 0.03, soybean lecithin 0.1, calcium carbonate 0.4, defoamer 0.15, the remainder being water;

[0065] Step (2)

[0066] The well-grown Bacillus polymyxa seed culture was inoculated into the above fermentation medium at a volume of 10% of the fermentation tank volume after inoculation. Before inoculation, the following conditions were controlled in the fermentation tank: aeration ratio of 0.6 vvm, tank pressure of 0.04 MPa-0.06 MPa, and rotation speed of 200-500 rpm. The dissolved oxygen was controlled at 30%-40% by adjusting the rotation speed.

[0067] Step (3)

[0068] The temperature was controlled at 30℃ throughout the process, and the pH was left to stand naturally (after inoculation of the fermenter medium, cultivation was carried out under the above conditions without intervention on pH changes) for 60 hours to obtain polymyxin B fermentation broth; the final fermentation broth was analyzed by high-performance liquid chromatography, and the elution peak and yield of polymyxin B were as follows. Figure 2 As shown in Table 2:

[0069] Table 2

[0070]

[0071] From Table 1, Table 2, Figure 1 and Figure 2 It can be seen that, compared with the control example, Example 4, through formula supplementation, staged control of temperature and pH, and supplementation, precisely controlled the fermentation process, and the fermentation unit increased from 2.057 g / L in the control example to 2.662 g / L, an increase of 29.42%. Among them, supplementation of feed 2 significantly increased the content of effective component B1, raising the content of effective component to 75.28%. Compared with the control example, the content of effective substances in the final fermentation broth increased by 12.32%, and the quality of the final fermentation broth was significantly improved.

[0072] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing fermentation broth that increases the content of the effective component of polymyxin B, characterized in that, Includes the following steps: S1. Inoculate the well-grown Bacillus polymyxa seed culture into the fermentation medium at a volume of 10% of the fermentation tank volume after inoculation. Before inoculation, the aeration rate is 0.6 vvm, the tank pressure is 0.04MPa-0.06MPa, the rotation speed is 200-500rpm, and the dissolved oxygen content is 30%≤40%. S2. During fermentation from 0h to 12h, control the temperature inside the fermenter at 30-32℃, and add feed one to control the pH inside the fermenter at 6.5-6.7; during fermentation from 13h to 60h, control the temperature inside the fermenter at 27-29℃, add feed two at 0.0001-0.0003 times the volume of fermentation liquid every hour, and add feed one to control the pH inside the fermenter at 6.1-6.

3. The second feed is a sterilized carnosine solution with a concentration of 5% (w / v).

2. The method for preparing fermentation broth to increase the content of effective components of polymyxin B according to claim 1, characterized in that, Also includes: S3. After fermentation for 30 hours, feed 0.002-0.004 times the fermentation volume of feed 3 at a constant rate every hour, and culture for 60 hours.

3. The method for preparing fermentation broth to increase the content of effective components of polymyxin B according to claim 1, characterized in that, The fermentation medium in step S1 is composed of the following components by weight per 100 mL: 6.0 g-8.0 g whole wheat flour, 0.01 g-0.02 g α-amylase, 0.3 g-0.5 g glucose, 1.5 g-2.5 g low-temperature soybean flour, 0.4 g-0.6 g corn steep liquor, 0.6 g-0.8 g ammonium sulfate, 0.025 g-0.035 g potassium dihydrogen phosphate, 0.05 g-0.15 g soybean lecithin, 0.3 g-0.5 g calcium carbonate, 0.1 g-0.2 g defoamer, and the remainder is water.

4. The method for preparing fermentation broth to increase the content of effective components of polymyxin B according to claim 1, characterized in that: The feed is ammonia water with a concentration of 20%.

5. The method for preparing fermentation broth to increase the content of effective components of polymyxin B according to claim 2, characterized in that: The third feed is a sterilized 50% (w / v) glucose solution.

6. The method for preparing fermentation broth to increase the content of effective components of polymyxin B according to claim 3, characterized in that: The fermentation medium in step S1 is composed of the following components by weight per 100 mL: 7.0 g whole wheat flour, 0.015 g α-amylase, 0.4 g glucose, 2.0 g low-temperature soybean flour, 0.5 g corn steep liquor, 0.7 g ammonium sulfate, 0.03 g potassium dihydrogen phosphate, 0.1 g soybean lecithin, 0.4 g calcium carbonate, 0.15 g defoamer, and the remainder is water.

7. The method for preparing fermentation broth to increase the content of effective components of polymyxin B according to claim 1, characterized in that: During the fermentation period from 0h to 12h, the temperature inside the fermentation tank is controlled at 31℃; during the fermentation period from 13h to 60h, the temperature inside the fermentation tank is controlled at 28℃.

8. The method for preparing fermentation broth to increase the content of effective components of polymyxin B according to claim 1, characterized in that: During the fermentation period of 0h-12h, the pH inside the fermenter was controlled at 6.6; during the fermentation period of 13h-60h, the pH inside the fermenter was controlled at 6.2.

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