Vancomycin fermentation system and fermentation method

Abstract

The invention provides a vancomycin fermentation system and a vancomycin fermentation method, and belongs to the technical field of microbial fermentation. The vancomycin fermentation system comprises a basic culture medium and a fed-batch culture medium, the basic culture medium is prepared from 10-30 g / L of glucose, 5-15 g / L of peptone, 2-8 g / L of yeast extract, 1-3 g / L of monopotassium phosphate and 0.5-1.5 g / L of magnesium sulfate; the fed-batch culture medium is a glucose-peptone mixed solution, and comprises 500g / L of glucose and 250-1000g / L of peptone; in the fermentation process, a fed-batch culture medium is regularly added. The fermentation capacity of amycolatopsis orientalis can be effectively improved, the vancomycin yield is increased, and the content of impurity-demethylated vancomycin in fermentation liquor is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and in particular relates to a vancomycin fermentation system and a fermentation method. 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 methicillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative Staphylococci (MRCNS). Therefore, vancomycin is widely used in the clinic to treat serious Gram-positive bacterial infections, especially those that are resistant to penicillin and other antibiotics.

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

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

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0007] The present invention also provides a vancomycin fermentation method based on the fermentation system, wherein the basal culture medium is added into a fermentation tank, Amycolatopsis orientalis is inoculated for fermentation, and the feed culture medium is added during the fermentation process.

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

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

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

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

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

[0013] (1) Improvement of fermentation efficiency: Based on the batch fermentation process, the present invention adds a feeding process of a glucose-peptone mixed solution, which can effectively improve the fermentation capacity of Amycolatopsis orientalis, 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 yield of the product, 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 of the present invention has higher quality and lower impurity content.

[0015] (3) Improving product quality stability: The present invention adopts a regular feeding method for fermentation, which can better control various parameters of the fermentation process, thereby making the yield and quality of vancomycin more stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a high performance liquid chromatogram of the supernatant obtained by the vancomycin fermentation method in Example 1;

[0017] Figure 2 This is a high performance liquid chromatogram of the supernatant obtained by the vancomycin fermentation method in Example 2;

[0018] Figure 3 This is a high performance liquid chromatogram of the supernatant obtained by the vancomycin fermentation method in Example 3;

[0019] Figure 4 This is a high performance liquid chromatogram of the supernatant prepared by the vancomycin fermentation method in Comparative Example 1. DETAILED DESCRIPTION

[0020] The present invention provides a vancomycin fermentation system, including a basal culture medium and a feed culture medium. The basal culture medium of the present invention is 10-30 g / L of glucose, 5-15 g / L of peptone, 2-8 g / L of yeast extract, 1-3 g / L of potassium dihydrogen phosphate, and 0.5-1.5 g / L of magnesium sulfate; more preferably, 20 g / L of glucose, 10 g / L of peptone, 5 g / L of yeast extract, 2 g / L of potassium dihydrogen phosphate, and 1 g / L of magnesium sulfate; more preferably, after the medium is prepared, it is sterilized at 121°C for 20 min. The feed culture medium of the present invention is a glucose-peptone mixture, 500 g / L of glucose, 250-1000 g / L of peptone, and further 500 g / L of peptone, 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 fermentation system, wherein the basal culture medium is added into a fermentation tank, Amycolatopsis orientalis is inoculated for fermentation, and the feed culture medium is added during the fermentation process.

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

[0023] In the present invention, preferably after 24 hours of fermentation, feed medium is added once every 10-14 hours, and the addition amount is 30-50 mL per L of basal medium; further preferably, feed medium is added once every 12 hours, and the addition amount is 40 mL per L of basal medium. As an implementable embodiment, taking a 5L fermentor as an example, 3L of basal medium is added for vancomycin fermentation, and 120mL of feed 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, more preferably 33°C; the dissolved oxygen is preferably 20%-25% before 24 hours, more preferably 20%; the dissolved oxygen is preferably 30%-50% after 24 hours, more preferably 40%; the rotation speed is preferably 200-800rpm, more preferably 500rpm.

[0025] In the present invention, preferably the fermentation lasts for 140-160 hours, more preferably 144 hours.

[0026] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0027] Example 1

[0028] A vancomycin fermentation method, comprising the following steps:

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

[0030] (2) The preserved Amycolatopsis orientalis was inoculated into a fermentation tank at an inoculation amount of 5% (v / v), and batch fermentation was started for 144 hours.

[0031] (3) During the fermentation process, feed medium was added to the fermenter every 12 hours after 24 hours. The amount added each time was 120 mL. The composition of the feed medium was: 500 g / L glucose and 500 g / L peptone.

[0032] Example 2

[0033] A vancomycin fermentation method comprises the following steps: the difference from Example 1 is that the composition of the feed medium is: 500 g / L glucose and 250 g / L peptone.

[0034] Example 3

[0035] A vancomycin fermentation method comprises the following steps: the difference from Example 1 is that the composition of the feed medium is: 500 g / L glucose and 1000 g / L peptone.

[0036] Comparative Example 1

[0037] A method for fermenting and purifying vancomycin comprises the following steps: the method is different from Example 1 in that only steps (1) and (2) are performed.

[0038] Fermentation broth treatment method:

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

[0040] Mobile phase preparation:

[0041] 1. Triethylamine buffer: add 10 mL triethylamine to 5000 mL ultrapure water, adjust pH to 3.2 with phosphoric acid; 2. Mixed solution: acetonitrile: tetrahydrofuran = 49 mL: 7 mL;

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

[0043] 4. Mobile phase B: triethylamine buffer: mixed solution = 40mL:40mL;

[0044] Testing conditions:

[0045] Flow rate: 1.5 mL;

[0046] Wavelength: 280nm;

[0047] Column temperature: 25°C;

[0048] Detection time: 35min;

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

[0050] Content calculation method:

[0051]

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

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

[0054] N--dilution multiple;

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

[0056] Demethylation calculation method:

[0057]

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

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

[0060] N--dilution multiple;

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

[0062] Demethylation ratio:

[0063]

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

[0065]

[0066]

[0067] From Table 1 and Figure 1-4 It can be seen that adding feed culture medium during the fermentation process by feeding can control the concentration of carbon source and nitrogen source in the fermentation process, reduce the influence of glucose effect and the external environment of the culture medium on the fermentation process, effectively improve the fermentation capacity of Amycolatopsis orientalis, thereby increasing the production of vancomycin, and can also effectively reduce the content of impurity demethylvancomycin in the fermentation broth.

[0068] Example 4

[0069] A vancomycin fermentation method, comprising the following steps:

[0070] (1) Prepare a 5L fermentation tank 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. Sterilization conditions are 121℃, 20min. After sterilization, set the fermentation tank temperature to 30℃, control the dissolved oxygen at 25% before 24h, and control the dissolved oxygen at 30% after 24h, and stir at 200rpm.

[0071] (2) The preserved Amycolatopsis orientalis was inoculated into a fermentation tank at an inoculum amount of 2% (v / v), and batch fermentation was started for 140 hours.

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

[0073] Example 5

[0074] A vancomycin fermentation method, comprising the following steps:

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

[0076] (2) The preserved Amycolatopsis orientalis was inoculated into a fermentation tank at an inoculum amount of 10% (v / v), and batch fermentation was started for 160 hours.

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

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A vancomycin fermentation system, characterized in that: The invention comprises a basal culture medium and a supplementary culture medium; the basal culture medium comprises 10-30 g / L glucose, 5-15 g / L peptone, 2-8 g / L yeast extract, 1-3 g / L potassium dihydrogen phosphate and 0.5-1.5 g / L magnesium sulfate; the supplementary culture medium comprises a glucose-peptone mixture, comprising 500 g / L glucose and 250-1000 g / L peptone.

2. A vancomycin fermentation method based on the fermentation system of claim 1, characterized in that: The basic culture medium is added into a fermentation tank, and Amycolatopsis orientalis is inoculated for fermentation, and the feed culture medium is added during the fermentation process.

3. The vancomycin fermentation method according to claim 2, characterized in that: The inoculation amount of Amycolatopsis orientalis is 2%-10%.

4. The vancomycin fermentation method according to claim 2, characterized in that: After 24 hours of fermentation, feed medium was added every 10-14 hours, and the added amount was 30-50 mL per L of basal medium.

5. The vancomycin fermentation method according to claim 2, characterized in that: During the fermentation process, the temperature is 30-35° C., the dissolved oxygen is 20%-25% before 24 hours, and 30%-50% after 24 hours, and the rotation speed is 200-800 rpm.

6. The vancomycin fermentation method according to claim 2, characterized in that: The fermentation lasted 140-160 h.

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