A method for increasing polymyxin b3 content in polymyxin b fermentation product

Abstract

This invention belongs to the field of bio-fermentation technology, and specifically relates to a method for increasing the polymyxin B3 content in polymyxin B fermentation products. Specifically, in a fed-batch fermentation method, a butyric acid solution is intermittently added to the fermentation broth after at least 12 hours of fermentation. The method provided by this invention can effectively increase the polymyxin B3 content in the fermentation broth to above 0.6 g / L.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, and specifically relates to a method for increasing the polymyxin B3 content in polymyxin B fermentation products. Background Technology

[0002] Polymyxin B is a cyclic peptide antibiotic produced by *Bacillus polymyxa*, which has a potent bactericidal effect against Gram-negative bacilli. Polymyxin B achieves its bactericidal effect by altering the permeability of the bacterial cell membrane.

[0003] With the widespread use of antibiotics, the problem of drug resistance in Gram-negative bacteria has become prominent. Polymyxin B is considered the last line of defense in treating infections caused by Gram-negative bacteria.

[0004] Polymyxin B is a multi-component substance comprising B1, B2, B3, and B1-I. Its structural formula is shown in general formula (1) and Table 1:

[0005]

[0006] Table 1

[0007] B1 <![CDATA[-CH2CH3]]> <![CDATA[-CH3]]> -H <![CDATA[-CH3]]> B2 <![CDATA[-CH3]]> <![CDATA[-CH3]]> -H <![CDATA[-CH3]]> B3 <![CDATA[-CH2CH3]]> -H -H <![CDATA[-CH3]]> B1-1 <![CDATA[-CH2CH3]]> <![CDATA[-CH3]]> <![CDATA[-CH3]]> -H

[0008] Existing literature contains limited research on polymyxin B3. Although there are relevant quality requirements for the components of polymyxins both domestically and internationally, each component still affects the quality and efficacy of the drug to some extent. Therefore, obtaining high-content polymyxin B3 is of great significance. Attached Figure Description

[0009] Figure 1 The image shown is a chromatogram of the fermentation broth from Example 1 of the present invention, as described in a specific embodiment.

[0010] Figure 2 The image shown is a chromatogram of the fermentation broth of Comparative Example 1 in a specific embodiment of the present invention. Summary of the Invention

[0011] In order to overcome the shortcomings of the prior art, the technical problem to be solved by the present invention is to provide a method that can effectively increase the polymyxin B3 content in polymyxin B fermentation products.

[0012] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: a method for increasing the polymyxin B3 content in polymyxin B fermentation products, wherein, under the fermentation mode of fed-batch fermentation, after fermentation culture for at least 12 hours, the feed butyric acid solution is added to the fermentation broth in an intermittent feeding manner.

[0013] The beneficial effects of this invention are as follows: Polymyxin B3 is a compound composed of a cyclic decapeptide and a fatty acid linked by peptide bonds. This invention involves adding butyric acid as a feedstock to the fermentation broth during the fermentation process. Since butyric acid is a precursor for fatty acid synthesis, as a small molecule, it is easily absorbed and utilized by the organism (fermentation strain), thereby participating in the synthesis of butyryl-CoA and entering the fatty acid synthesis pathway. Experiments have shown that adding an appropriate amount of butyric acid can effectively increase the fatty acid content in the organism, thereby increasing the polymyxin B3 content in the fermentation broth to over 0.6 g / L. Detailed Implementation

[0014] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments.

[0015] A method for increasing the polymyxin B3 content in polymyxin B fermentation products involves, in a fed-batch fermentation mode, adding a feed butyric acid solution to the fermentation broth intermittently after at least 12 hours of fermentation culture.

[0016] In this process, butyric acid solution is added to the fermentation broth intermittently after at least 12 hours of fermentation. Preferably, it is added in a single batch after 12 hours of fermentation. Butyric acid, as a precursor to fatty acid synthesis, possesses high bioavailability and utilization. It can synthesize butyryl-CoA via the butyrate-CoA pathway and enter the fatty acid synthesis pathway, thereby promoting the fatty acid content in the fermentation strain and increasing the polymyxin B3 content in the fermentation broth. Experiments have shown that adding an appropriate amount of butyric acid solution to the fermentation broth can increase the polymyxin B3 content to over 0.6 g / L.

[0017] Preferably, the concentration of the butyric acid solution is 15-25 wt%, more preferably 20 wt%. The amount of the butyric acid solution added is 0.05-0.15 (v / v)%, preferably 0.1 (v / v)%.

[0018] The fermentation strain is *P. polymyxa*.

[0019] Specifically, the method for increasing the polymyxin B3 content in the polymyxin B fermentation product includes the following steps:

[0020] S1. Cultivate the fermentation strain under seed culture until the seed matures to obtain mature seed liquid;

[0021] S2. Ferment the mature seed liquid for at least 12 hours, then add butyric acid to the fermentation liquid and continue fermentation for 38-44 hours.

[0022] In one embodiment, the seed culture is as follows: a seed culture medium is prepared, and the tank temperature is controlled at 28-30℃, the tank pressure at 0.03-0.05MPa, the stirring speed at 150-200rpm, and the aeration ratio at 0.7-1.0vvm. After inoculating the fermentation strain, the culture is carried out for 10-13 hours. The pH of the seed liquid drops to 6.0-6.2, and the mycelium of the fermentation strain is found to be evenly distributed under a microscope, thus obtaining a mature seed liquid.

[0023] Preferably, the seed culture medium comprises the following components by weight percentage: 8-9% wheat flour (food-grade refined wheat flour, purchased from Yihai Kerry Oils & Grains Foodstuffs Co., Ltd.), 0.2-0.3% yeast extract (purchased from Beijing Hongrun Baoshun Technology Co., Ltd.), 0.013-0.016% dipotassium hydrogen phosphate, 0.3-0.4% calcium carbonate, 0.40-0.50% ammonium sulfate, 0.04-0.06% defoamer (GPE, purchased from Jiangsu Lihong Technology Development Co., Ltd.), with the balance being water. More preferably, the seed culture medium is prepared from the following components by weight percentage: 8% wheat flour, 0.3% yeast extract, 0.015% dipotassium hydrogen phosphate, 0.3% calcium carbonate, 0.45% ammonium sulfate, 0.04% defoamer, with the balance being water.

[0024] In one embodiment, the fermentation culture is as follows: a fermentation culture medium is prepared, and the tank temperature is controlled at 28-30°C, the tank pressure at 0.03-0.05 MPa, the stirring speed at 150-600 rpm, the aeration ratio at 0.7-1.5 vvm, and the dissolved oxygen value at ≥40%.

[0025] Preferably, the fermentation medium comprises the following components by weight percentage: 10-11% wheat flour, 0.7-0.9% cottonseed meal (purchased from Jiangsu Jiurui Technology Co., Ltd.), 0.013-0.016% dipotassium hydrogen phosphate, 0.3-0.4% calcium carbonate, 0.4-0.50% ammonium sulfate, 0.03-0.06% defoamer (GPE, purchased from Jiangsu Lihong Technology Development Co., Ltd.), and the balance being water. More preferably, the fermentation medium is prepared from the following components by weight percentage: 10% wheat flour, 0.8% cottonseed meal, 0.015% dipotassium hydrogen phosphate, 0.3% calcium carbonate, 0.45% ammonium sulfate, 0.03% defoamer, and the balance being water.

[0026] Preferably, the inoculation amount of the mature seed liquid is 7.5–15 (v / v)%.

[0027] Example 1

[0028] A method for increasing the polymyxin B3 content in polymyxin B fermentation products includes the following steps:

[0029] S1. Seed culture: Prepare seed culture medium, control the tank temperature at 29℃, the tank pressure at 0.04MPa, stir at 150-200rpm, and the aeration ratio at 0.7-1.0vvm. Inoculate with polymyxin B-producing Bacillus polymyxin for culture. Control the dissolved oxygen (DO) value on the seed tank to be ≥40%. The culture period is 10-13h. When the pH drops to 6.0-6.2, the seeds are considered mature when the mycelium is evenly distributed under a microscope.

[0030] The seed culture medium used consists of: 8% flour, 0.3% yeast extract, 0.015% dipotassium hydrogen phosphate, 0.3% calcium carbonate, 0.45% ammonium sulfate, 0.04% defoamer, and the remainder is water.

[0031] S2. Inoculate the mature polymyxin B seed culture at a rate of 15% into a fermenter containing fermentation medium and culture it. Control the temperature of the fermenter to 29℃, the pressure to 0.04MPa, the stirring speed to 150-600rpm, the aeration ratio to 0.7-1.5vvm, and the dissolved oxygen (DO) in the fermenter to be ≥40%.

[0032] The fermentation medium consists of the following ingredients: 10% wheat flour, 0.8% cottonseed meal, 0.015% dipotassium hydrogen phosphate, 0.3% calcium carbonate, 0.45% ammonium sulfate, 0.03% defoamer, and the remainder is water.

[0033] S3. After 12 hours of fermentation, add 0.1% n-butyric acid solution to the fermentation broth. End fermentation after 42 hours.

[0034] After testing (such as) Figure 1 For specific testing methods, please refer to the Chinese Pharmacopoeia for the high performance liquid chromatography method of polymyxin B sulfate. The average content of polymyxin B3 in the fermentation broth was 0.624 g / L.

[0035] Example 2

[0036] A method for increasing the polymyxin B3 content in polymyxin B fermentation products includes the following steps:

[0037] S1. Seed culture: Prepare seed culture medium, control the tank temperature at 29℃, the tank pressure at 0.04MPa, stir at 150-200rpm, and the aeration ratio at 0.7-1.0vvm. Inoculate with polymyxin B-producing Bacillus polymyxin for culture. Control the dissolved oxygen (DO) value on the seed tank to be ≥40%. The culture period is 10-13h. When the pH drops to 6.0-6.2, the seeds are considered mature when the mycelium is evenly distributed under a microscope.

[0038] The seed culture medium used consists of: 8% flour, 0.3% yeast extract, 0.015% dipotassium hydrogen phosphate, 0.3% calcium carbonate, 0.45% ammonium sulfate, 0.04% defoamer, and the remainder is water.

[0039] S2. Inoculate the mature polymyxin B seed culture at a rate of 15% into a fermenter containing fermentation medium and culture it. Control the temperature of the fermenter to 29℃, the pressure to 0.04MPa, the stirring speed to 150-600rpm, the aeration ratio to 0.7-1.5vvm, and the dissolved oxygen (DO) in the fermenter to be ≥40%.

[0040] The fermentation medium consists of the following ingredients: 10% wheat flour, 0.8% cottonseed meal, 0.015% dipotassium hydrogen phosphate, 0.3% calcium carbonate, 0.45% ammonium sulfate, 0.03% defoamer, and the remainder is water.

[0041] S3. After 12 hours of fermentation, add 0.1% n-butyric acid solution to the fermentation broth. End fermentation after 42 hours.

[0042] The average content of polymyxin B3 in the fermentation broth was found to be 0.607 g / L.

[0043] Example 3

[0044] A method for increasing the polymyxin B3 content in polymyxin B fermentation products includes the following steps:

[0045] S1. Seed culture: Prepare seed culture medium, control the tank temperature at 29℃, the tank pressure at 0.04MPa, stir at 150-200rpm, and the aeration ratio at 0.7-1.0vvm. Inoculate with polymyxin B-producing Bacillus polymyxin for culture. Control the dissolved oxygen (DO) value on the seed tank to be ≥40%. The culture period is 10-13h. When the pH drops to 6.0-6.2, the seeds are considered mature when the mycelium is evenly distributed under a microscope.

[0046] The seed culture medium used consists of: 8% flour, 0.3% yeast extract, 0.015% dipotassium hydrogen phosphate, 0.3% calcium carbonate, 0.45% ammonium sulfate, 0.04% defoamer, and the remainder is water.

[0047] S2. Inoculate the mature polymyxin B seed culture at a rate of 15% into a fermenter containing fermentation medium and culture it. Control the temperature of the fermenter to 29℃, the pressure to 0.04MPa, the stirring speed to 150-600rpm, the aeration ratio to 0.7-1.5vvm, and the dissolved oxygen (DO) in the fermenter to be ≥40%.

[0048] The fermentation medium consists of the following ingredients: 10% wheat flour, 0.8% cottonseed meal, 0.015% dipotassium hydrogen phosphate, 0.3% calcium carbonate, 0.45% ammonium sulfate, 0.03% defoamer, and the remainder is water.

[0049] S3. After 12 hours of fermentation, add 0.1% n-butyric acid solution to the fermentation broth. End fermentation after 42 hours.

[0050] The average content of polymyxin B3 in the fermentation broth was found to be 0.647 g / L.

[0051] Comparative Example 1

[0052] A method for increasing the polymyxin B3 content in polymyxin B fermentation products, differing from Example 1, involves replacing the butyric acid solution with an equal volume of water during fermentation culture 12 hours. (See [link to example 1]). Figure 2 The average content of polymyxin B3 in the fermentation broth was 0.143 g / L.

[0053] It should be noted that Examples 1 to 3 are essentially three repeated experiments under the same production conditions, and all parameters fluctuated within the aforementioned range during fermentation. For example, the dissolved oxygen (DO) in the fermenter was maintained at ≥40% by controlling the stirring rate and aeration ratio during fermentation.

[0054] Experimental Example

[0055] A control group and experimental groups 1-5 were set up. Experimental groups 1-5 were fermented according to the method of Example 1, while the control group was fermented according to the method of Comparative Example 1. Specific results are shown in Table 2.

[0056] Table 2

[0057] control group 0.136 Experimental group 1 0.602 Experimental group 2 0.628 Experimental group 3 0.613 Experimental group 4 0.669 Experimental group 5 0.635

[0058] As can be seen from Table 1, adding butyric acid during the fermentation process can significantly increase the content of polymyxin B3 in the fermentation broth.

[0059] In summary, polymyxin B3 is a compound composed of a cyclic decapeptide and a fatty acid linked by peptide bonds. This invention involves adding butyric acid as a feedstock to the fermentation broth during fermentation. Since butyric acid is a precursor to fatty acid synthesis, as a small molecule, it is readily absorbed and utilized by the organism (fermentation strain), thereby participating in the synthesis of butyryl-CoA and entering the fatty acid synthesis pathway. Experiments have demonstrated that adding an appropriate amount of butyric acid can effectively increase the fatty acid content in the organism, thereby increasing the polymyxin B3 content in the fermentation broth to over 0.6 g / L.

[0060] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for increasing the polymyxin B3 content in polymyxin B fermentation products, characterized in that, In the fed-batch fermentation method, after fermentation culture for at least 12 hours, the feed butyric acid solution is added to the fermentation broth in an intermittent feeding manner; the fermentation strain is Bacillus polymyxa. The concentration of the butyric acid solution is 20 wt%; The amount of butyric acid solution added is 0.1 v / v.

2. The method according to claim 1, characterized in that, Includes the following steps: S1. Cultivate the fermentation strain under seed culture until the seed matures to obtain mature seed liquid; S2. Ferment the mature seed liquid for at least 12 hours, then add butyric acid to the fermentation liquid and continue fermentation for 38-44 hours.

3. The method according to claim 2, characterized in that, The seed culture is as follows: prepare seed culture medium, control tank temperature at 28~30℃, tank pressure at 0.03~0.05MPa, stirring speed at 150~200rpm, and aeration ratio at 0.7~1.0vvm. After inoculating with fermentation strains, culture for 10~13h until the pH of the seed liquid drops to 6.0~6.

2. Microscopic examination shows that the mycelium of the fermentation strains is evenly distributed, thus obtaining a mature seed liquid.

4. The method according to claim 3, characterized in that, The seed culture medium comprises the following components in weight percentage: The ingredients are: flour 8-9%, yeast extract 0.2-0.3%, dipotassium hydrogen phosphate 0.013-0.016%, calcium carbonate 0.3-0.4%, ammonium sulfate 0.40-0.50%, defoamer 0.04-0.06%, and the remainder is water.

5. The method according to claim 2, characterized in that, The fermentation culture is as follows: prepare fermentation culture medium, control tank temperature at 28~30℃, tank pressure at 0.03~0.05MPa, stirring speed at 150~600rpm, aeration ratio at 0.7~1.5vvm, and dissolved oxygen value at ≥40%.

6. The method according to claim 5, characterized in that, The fermentation medium comprises the following components by weight percentage: The ingredients are: flour 10-11%, cottonseed flour 0.7-0.9%, dipotassium hydrogen phosphate 0.013-0.016%, calcium carbonate 0.3-0.4%, ammonium sulfate 0.4-0.50%, defoamer 0.03-0.06%, and the remainder is water.

7. The method according to claim 2, characterized in that, The inoculation volume of the mature seed solution is 7.5~15 v / v.

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