A fermentation system and method for vancomycin

Abstract

The present invention provides a fermentation system and a fermentation method for vancomycin, belonging to the technical field of microbial fermentation. The vancomycin fermentation system of the present invention comprises a basal medium and a feeding medium; the basal medium is glucose 10 - 30 g / L, peptone 5 - 15 / L, yeast extract 2 - 8 g / L, potassium dihydrogen phosphate 1 - 3 g / L, magnesium sulfate 0.5 - 1.5 g / L; the feeding medium is a glucose peptone mixture, glucose 500 g / L, peptone 250 - 1000 g / L; during the fermentation process, the feeding medium is added regularly. The present invention can effectively improve the fermentation ability of Amycolatopsis orientalis, increase the yield of vancomycin, and reduce the content of impurities - demethylvancomycin in the fermentation broth.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation system and a fermentation method for vancomycin. Background Art

[0002] Vancomycin is a glycopeptide antibiotic produced by Amycolatopsis orientalis under fermentation conditions. It has strong antibacterial activity against a variety of drug-resistant bacteria, especially has a unique bactericidal effect on methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (MRCNS). Therefore, vancomycin is widely used clinically to treat severe Gram-positive bacterial infections, especially for those bacterial infections resistant to penicillin and other antibiotics.

[0003] Currently, the production of vancomycin mainly adopts the batch fermentation process. This process has been widely used in industrial production and has achieved certain success. However, due to the complex biosynthesis process of vancomycin and numerous influencing factors, such as the glucose effect and the external environment of the culture medium, how to optimize the fermentation conditions and improve the yield and quality of vancomycin remains an important research topic. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a fermentation system and a fermentation method for vancomycin, which can effectively improve the fermentation ability of Amycolatopsis orientalis, increase the yield of vancomycin, and reduce the content of impurity demethylvancomycin in the fermentation broth.

[0005] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] A vancomycin fermentation system includes a basal medium and a feeding medium; the basal medium is glucose 10 - 30 g / L, peptone 5 - 15 g / L, yeast extract 2 - 8 g / L, potassium dihydrogen phosphate 1 - 3 g / L, magnesium sulfate 0.5 - 1.5 g / L; the feeding medium is a glucose peptone mixture, glucose 500 g / L, peptone 250 - 1000 g / L.

[0007] The present invention also provides a vancomycin fermentation method based on the above fermentation system. Add the basal medium into a fermenter, inoculate Amycolatopsis orientalis for fermentation, and add the feeding medium during the fermentation process.

[0008] Preferably, the inoculation amount of Amycolatopsis orientalis is 2% - 10%.

[0009] Preferably, after 24 h of fermentation, the feeding medium is added once every 10 - 14 h, and the addition amount is 30 - 50 mL per L of the basal medium.

[0010] Preferably, during the fermentation process, the temperature is 30 - 35°C, the dissolved oxygen is 20% - 25% before 24 h, the dissolved oxygen is 30% - 50% after 24 h, and the rotation speed is 200 - 800 rpm.

[0011] Preferably, the fermentation lasts for 140 - 160 h.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] (1) Improving the fermentation efficiency: Based on the batch fermentation process, the present invention adds a feeding process of a glucose peptone mixed solution. In this way, the fermentation ability of Amycolatopsis orientalis can be effectively improved, thereby increasing the yield of vancomycin. Compared with the traditional batch fermentation process, the fermentation efficiency of the present invention is higher, which can not only increase the product yield but also reduce the production cost.

[0014] (2) Reducing the impurity content: The fermentation method of the present invention can not only increase the yield of vancomycin but also effectively reduce the content of the impurity demethylvancomycin in the fermentation broth. This means that the product quality of the present invention is higher and the impurity content is lower.

[0015] (3) Improving the product quality stability: The present invention uses a method of regular feeding for fermentation, which can better control various parameters of the fermentation process, so that the yield and quality of vancomycin are more stable. Description of the Drawings

[0016] Figure 1 It is the high - performance liquid chromatography diagram of the supernatant obtained by the vancomycin fermentation method in Example 1;

[0017] Figure 2 It is the high - performance liquid chromatography diagram of the supernatant obtained by the vancomycin fermentation method in Example 2;

[0018] Figure 3 It is the high - performance liquid chromatography diagram of the supernatant obtained by the vancomycin fermentation method in Example 3;

[0019] Figure 4 It is the high - performance liquid chromatography diagram of the supernatant obtained by the vancomycin fermentation method in Comparative Example 1. Detailed Embodiments

[0020] The present invention provides a vancomycin fermentation system, including a basal medium and a feeding medium. The basal medium of the present invention contains glucose at 10 - 30 g / L, peptone at 5 - 15 g / L, yeast extract at 2 - 8 g / L, potassium dihydrogen phosphate at 1 - 3 g / L, and magnesium sulfate at 0.5 - 1.5 g / L; further preferably, glucose is 20 g / L, peptone is 10 g / L, yeast extract is 5 g / L, potassium dihydrogen phosphate is 2 g / L, and magnesium sulfate is 1 g / L; more preferably, after the medium is prepared, it is sterilized at 121 °C for 20 min. The feeding medium of the present invention is a glucose-peptone mixture, with glucose at 500 g / L and peptone at 250 - 1000 g / L, and further preferably peptone at 500 g / L, that is, the concentration ratio of glucose to peptone is 1:1.

[0021] The present invention also provides a vancomycin fermentation method based on the above fermentation system. Add the basal medium into a fermenter, inoculate Amycolatopsis orientalis for fermentation, and add the feeding medium during the fermentation process.

[0022] In the present invention, the inoculation amount of Amycolatopsis orientalis is preferably 2% - 10% (v / v), and further preferably 5% (v / v).

[0023] In the present invention, it is preferred that after 24 h of fermentation, the feeding medium is added once every 10 - 14 h, and the addition amount is 30 - 50 mL per L of the basal medium; more preferably, the feeding medium is added once every 12 h, and the addition amount is 40 mL per L of the basal medium. As an implementable mode, taking a 5 L fermenter as an example, 3 L of the basal medium is added for vancomycin fermentation, and 120 mL of the feeding medium is added each time during the fermentation process.

[0024] In the present invention, during the fermentation process, the temperature is preferably 30 - 35 °C, and further preferably 33 °C; the dissolved oxygen is preferably 20% - 25% before 24 h, and further preferably 20%; the dissolved oxygen is preferably 30% - 50% after 24 h, and further preferably 40%; the rotation speed is preferably 200 - 800 rpm, and further preferably 500 rpm.

[0025] In the present invention, the fermentation is preferably continued for 140 - 160 h, and further preferably 144 h.

[0026] The following combines examples to detail the technical solutions provided by the present invention, but they cannot be construed as limiting the protection scope of the present invention.

[0027] Example 1

[0028] A vancomycin fermentation method, the steps are as follows:

[0029] (1) Prepare a 5L fermenter, add 3L of basal medium. The composition of the basal medium is: glucose 20g / L, peptone 10g / L, yeast extract 5g / L, potassium dihydrogen phosphate 2g / L, magnesium sulfate 1g / L. Sterilize at 121°C for 20 minutes. After sterilization, set the temperature of the fermenter at 33°C, control the dissolved oxygen at 20% 24 hours before, and control the dissolved oxygen at 40% 24 hours later, with an associated stirring speed of 500rpm.

[0030] (2) Inoculate the preserved Amycolatopsis orientalis into the fermenter, with an inoculation amount of 5% (v / v). Start batch fermentation, and the fermentation time is 144 hours.

[0031] (3) During the fermentation process, every 12 hours after 24 hours, add the feeding medium to the fermenter, with each addition amount of 120mL. The composition of the feeding medium is: glucose 500g / L, peptone 500g / L.

[0032] Example 2

[0033] A method for vancomycin fermentation, the steps are as follows: Different from Example 1 in that the composition of the feeding medium is: glucose 500g / L, peptone 250g / L.

[0034] Example 3

[0035] A method for vancomycin fermentation, the steps are as follows: Different from Example 1 in that the composition of the feeding medium is: glucose 500g / L, peptone 1000g / L.

[0036] Comparative Example 1

[0037] A method for vancomycin fermentation and purification, the steps are as follows: Different from Example 1 in that only steps (1) and (2) are carried out.

[0038] Fermentation broth treatment method:

[0039] Weigh 15g of the fermentation broth from Examples 1 - 3 and Comparative Example 1 respectively, add 15mL of purified water, adjust the pH value to 10.0 - 10.3 with NaOH, centrifuge at 3500rpm for 10 minutes, and take the supernatant for liquid phase determination of the content. The high performance liquid detection method is as follows:

[0040] Mobile phase preparation:

[0041] 1. Triethylamine buffer solution: Add 10mL of triethylamine to 5000mL of ultrapure water, and adjust the pH to 3.2 with phosphoric acid; 2. Mixed solution: Acetonitrile: Tetrahydrofuran = 49mL:7mL;

[0042] 3. Mobile phase A: Triethylamine buffer solution: Mixed solution = 186mL:14mL;

[0043] 4. Mobile phase B: Triethylamine buffer: mixture = 40 mL:40 mL;

[0044] Detection conditions:

[0045] Flow rate: 1.5 mL;

[0046] Wavelength: 280 nm;

[0047] Column temperature: 25 °C;

[0048] Detection time: 35 min;

[0049] Chromatographic column: HpLc column 250 mm * 4.6 mm, 5 μm;

[0050] Content calculation method:

[0051]

[0052] C--VCM-B concentration of the standard solution;

[0053] --Peak area of VCM-B in the test solution;

[0054] N--Dilution factor;

[0055] --Peak area of VCM-B in the standard solution;

[0056] Demethylation calculation method:

[0057]

[0058] C--VCM-B concentration of the standard solution;

[0059] --Peak area of DM-VCM in the test solution;

[0060] N--Dilution factor;

[0061] --Peak area of VCM-B in the standard solution;

[0062] Demethylation ratio:

[0063]

[0064] Table 1 Effects of different feeding media on vancomycin fermentation yield and quality

[0065]

[0066]

[0067] As can be seen from Table 1 and Figures 1-4 Figures 1-4 It can be seen that by adding supplementary medium during the fermentation process in a fed-batch manner, the concentrations of carbon source and nitrogen source during the fermentation process can be controlled, the glucose effect and the influence of the external environment of the medium on the fermentation process can be reduced, the fermentation ability of Amycolatopsis orientalis can be effectively improved, thereby increasing the yield of vancomycin, and the content of impurities demethylvancomycin in the fermentation broth can also be effectively reduced.

[0068] Example 4

[0069] A method for fermenting vancomycin, the steps are as follows:

[0070] (1) Prepare a 5L fermenter and add 3L of basal medium. The composition of the basal medium is: glucose 10g / L, peptone 5g / L, yeast extract 2g / L, potassium dihydrogen phosphate 1g / L, magnesium sulfate 0.5g / L. The sterilization conditions are 121°C for 20 min. After sterilization, set the temperature of the fermenter at 30°C, control the dissolved oxygen at 25% 24 hours before, and control the dissolved oxygen at 30% 24 hours later, with a stirring speed of 200 rpm.

[0071] (2) Inoculate the preserved Amycolatopsis orientalis into the fermenter at an inoculum size of 2% (v / v), and start batch fermentation for 140 hours.

[0072] (3) During the fermentation process, 100 mL of supplementary medium is added to the fermenter every 12 hours after 24 hours. The composition of the supplementary medium is: glucose 500g / L, peptone 400g / L.

[0073] Example 5

[0074] A method for fermenting vancomycin, the steps are as follows:

[0075] (1) Prepare a 5L fermenter and add 3L of basal medium. The composition of the basal medium is: glucose 30g / L, peptone 15g / L, yeast extract 8g / L, potassium dihydrogen phosphate 3g / L, magnesium sulfate 1.5g / L. The sterilization conditions are 121°C for 20 min. After sterilization, set the temperature of the fermenter at 35°C, control the dissolved oxygen at 22% 24 hours before, and control the dissolved oxygen at 50% 24 hours later, with a stirring speed of 800 rpm.

[0076] (2) Inoculate the preserved Amycolatopsis orientalis into the fermenter at an inoculum size of 10% (v / v), and start batch fermentation for 160 hours.

[0077] (3) During the fermentation process, 150 mL of supplementary medium is added to the fermenter every 12 hours after 24 hours. The composition of the supplementary medium is: glucose 500g / L, peptone 800g / L.

[0078] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for vancomycin fermentation, characterized in that, Add the basal medium to the fermenter, inoculate Amycolatopsis orientalis for fermentation, and add the feeding medium during the fermentation process; The basal medium is composed of glucose at 10 - 30 g / L, peptone at 5 - 15 g / L, yeast extract at 2 - 8 g / L, potassium dihydrogen phosphate at 1 - 3 g / L, and magnesium sulfate at 0.5 - 1.5 g / L; the feeding medium is a glucose-peptone mixture, with glucose at 500 g / L and peptone at 250 - 1000 g / L; The inoculation amount of Amycolatopsis orientalis is 2% - 10%; After 24 h of fermentation, add the feeding medium once every 10 - 14 h, and the addition amount is 30 - 50 mL per L of the basal medium; During the fermentation process, the temperature is 30 - 35 °C, the dissolved oxygen is 20% - 25% before 24 h, 30% - 50% after 24 h, and the rotation speed is 200 - 800 rpm; the fermentation lasts for 140 - 160 h.

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