Method for improving conversion rate of betamethasone epoxy dehydrogenation product

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a method for improving the conversion rate of betamethasone epoxy dehydrogenation substances, which comprises the following steps: step 1, adding a culture medium into a fermentation tank, and sequentially carrying out heating sterilization, cooling, inoculation and strain culture; 2, after culture is completed, betamethasone epoxide and absolute ethyl alcohol are put in multiple times, the weight ratio of the betamethasone epoxide to the absolute ethyl alcohol is 10: (10-15), and after the conversion rate reaches the standard, the temperature is increased, and the mixture is put into a tank. According to the method, after heating sterilization, cooling, inoculation and strain culture are sequentially carried out, the betamethasone epoxide and the absolute ethyl alcohol are added in multiple times in a multiple-feeding mode, the conversion time is shortened, the conversion rate of the betamethasone epoxy dehydrogenation product is increased, and the method has the industrial application value.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, in particular to a method for improving the conversion rate of betamethasone epoxide dehydrogenation products. Background Art

[0002] The chemical name of betamethasone epoxy dehydrogenate is 9b,11b-epoxy-16b-methylpregna-1,4-diene-17a,21-diol-3,20-dione, which is an important intermediate for synthesizing glucocorticoids such as betamethasone, and its product has anti-inflammatory, anti-allergic, immunosuppressive and antiviral effects. The preparation of betamethasone epoxy dehydrogenate is usually to put the substrate betamethasone epoxy into the cultured fermentation liquid at one time for conversion, which has the problems of long conversion time and low conversion rate, greatly increases the production cost, and restricts the marketization of the product. In view of this, the present invention is specially proposed. Summary of the invention

[0003] The object of the present invention is to provide a method for improving the conversion rate of betamethasone epoxide dehydrogenation product, which can solve the above technical problems.

[0004] The present invention provides a method for improving the conversion rate of betamethasone epoxide dehydrogenate, comprising the following steps:

[0005] Step 1: Add the culture medium into the fermentation tank, and perform heating and sterilization, cooling, and inoculation in sequence to culture the bacteria;

[0006] Step 2: After the cultivation is completed, betamethasone epoxide and anhydrous ethanol are added in multiple times, and the weight ratio of betamethasone epoxide to anhydrous ethanol is 10: (10-15). After the conversion rate reaches the standard, the temperature is increased and the tank is released.

[0007] Preferably, in step 1, the bacterial species used is Arthrobacter simplex, with a preservation number of CPCC141432.

[0008] Preferably, the culture medium comprises, by weight: 90-110 parts of glucose, 60-80 parts of peptone, 60-80 parts of corn steep liquor, 4-6 parts of potassium dihydrogen phosphate and 10,000 parts of water.

[0009] Preferably, the culture medium comprises, by weight: 100 parts of glucose, 70 parts of peptone, 70 parts of corn steep liquor, 5 parts of potassium dihydrogen phosphate and 10,000 parts of water.

[0010] Preferably, in step 1, the temperature is raised to 121-130° C. and kept warm for 20-30 minutes.

[0011] Preferably, in step 1, the temperature is lowered to 29-31°C.

[0012] Preferably, in step 1, the bacterial strain is cultured for 12-24 hours.

[0013] Preferably, betamethasone epoxide and anhydrous ethanol are added in multiple times with an interval of 8-12 hours between each addition.

[0014] Preferably, the feeding times in step 2 is 4-6 times.

[0015] Preferably, in step 2, the temperature is raised to 80-90° C., kept warm for 50-60 minutes, and the strains are inactivated to ensure that the fermentation environment is not polluted. Specifically, the strains will continue to carry out metabolic activities and catalytic reactions during the fermentation process. When the conversion of betamethasone epoxy dehydrogenation reaches the expected conversion rate, the strains need to be inactivated in time to terminate the reaction and prevent excessive reaction from causing changes in the structure and content of the product, affecting the quality and yield of the product. If the strains are not inactivated, in subsequent separation, extraction and refining operations, the live strains may continue to grow, reproduce and metabolize to produce new metabolites, which will increase the difficulty of separation and purification, make the extraction and refining process of the target product more complicated, and reduce production efficiency. Moreover, the live strains may adhere to the product, affecting the purity and quality of the product. The strains used in the fermentation process may cause potential biological hazards to the environment and operators. If the inactivation treatment is not carried out, microbial contamination may occur when discharging the fermentation liquid or handling the discarded strains, which will have an adverse effect on the surrounding environment and personnel health. In addition, inactivated bacteria may contaminate production equipment and other products during subsequent operations, increasing the difficulty and cost of cleaning and disinfection.

[0016] Beneficial effects of the present invention:

[0017] The invention sequentially performs heating sterilization, cooling, inoculation, and bacterial culture, and then feeds betamethasone epoxide and anhydrous ethanol in multiple times by multiple feeding methods, thereby shortening the conversion time and improving the conversion rate of betamethasone epoxide dehydrogenate, and having greater industrial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 It is the HPLC conversion pattern of Example 1 in the present invention;

[0020] Figure 2 It is the HPLC conversion pattern of Example 2 in the present invention;

[0021] Figure 3 It is the HPLC conversion pattern of Example 3 in the present invention;

[0022] Figure 4 It is the HPLC conversion pattern of the comparative example in the present invention. DETAILED DESCRIPTION

[0023] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0024] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form also includes the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this description, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0025] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Embodiment 1:

[0027] 100 g glucose, 70 g peptone, 70 g corn steep liquor, 5 g potassium dihydrogen phosphate were added to the fermentation tank, 10 L drinking water was added, the temperature was raised to 121 ° C, kept warm for 30 min, cooled to 29 ° C, inoculated and cultured for 24 h, 100 g betamethasone epoxide and 150 g anhydrous ethanol were added, the conversion was about 8 h, sampling was performed, and the TLC was 85.3%, and the secondary feeding was performed, 100 g betamethasone epoxide and 150 g anhydrous ethanol were added, the conversion was about 8 h, and the TLC was detected. The conversion rate is about 90%; three feedings are performed, 100g of betamethasone epoxide and 150g of anhydrous ethanol are added, the conversion is about 10h, and the conversion rate is about 90% detected by TLC; four feedings are performed, 100g of betamethasone epoxide and 100g of anhydrous ethanol are added, the conversion is about 10h, and the conversion rate is about 90% detected by TLC; five feedings are performed, 100g of betamethasone epoxide and 100g of anhydrous ethanol are added, the conversion is about 12h, and the conversion rate is 93.1% detected by HPLC.

[0028] Embodiment 2:

[0029] 100 g glucose, 70 g peptone, 70 g corn steep liquor, 5 g potassium dihydrogen phosphate were added to the fermentation tank, 10 L drinking water was added, the temperature was raised to 130 ° C, kept warm for 20 min, cooled to 31 ° C, inoculated and cultured for 14 h, 100 g betamethasone epoxide and 150 g anhydrous ethanol were added, the conversion was about 8 h, sampling was performed, and the TLC was 85.2%, and the secondary feeding was performed, 100 g betamethasone epoxide and 150 g anhydrous ethanol were added, the conversion was about 10 h, and the TLC was detected. The conversion rate is about 90%; three feedings are performed, 100g of betamethasone epoxide and 150g of anhydrous ethanol are added, the conversion is about 10h, and the conversion rate is about 90% detected by TLC; four feedings are performed, 100g of betamethasone epoxide and 100g of anhydrous ethanol are added, the conversion is about 10h, and the conversion rate is about 90% detected by TLC; five feedings are performed, 100g of betamethasone epoxide and 100g of anhydrous ethanol are added, the conversion is about 12h, and the conversion rate is 92.2% detected by HPLC.

[0030] Embodiment 3:

[0031] 100 g glucose, 70 g peptone, 70 g corn steep liquor, 5 g potassium dihydrogen phosphate were added to the fermentation tank, and 10 L drinking water was added. The temperature was raised to 125 ° C, kept warm for 25 min, cooled to 30 ° C, inoculated and cultured for 18 h, 100 g betamethasone epoxide and 150 g anhydrous ethanol were added, and the conversion was about 8 h. Samples were taken and the TLC was 85.4%. Secondary feeding was performed, and 100 g betamethasone epoxide and 150 g anhydrous ethanol were added. The conversion was about 10 h. TLC detection was performed. The conversion rate is about 90%; three feedings are performed, 100g of betamethasone epoxide and 150g of anhydrous ethanol are added, the conversion is about 10h, and the conversion rate is about 90% detected by TLC; four feedings are performed, 100g of betamethasone epoxide and 100g of anhydrous ethanol are added, the conversion is about 10h, and the conversion rate is about 90% detected by TLC; five feedings are performed, 100g of betamethasone epoxide and 100g of anhydrous ethanol are added, the conversion is about 12h, and the conversion rate is 95.1% detected by HPLC.

[0032] Comparative Example

[0033] 100 g of glucose, 70 g of peptone, 70 g of corn steep liquor, and 5 g of potassium dihydrogen phosphate were added to a fermentation tank, and 10 L of drinking water was added. The temperature was raised to 130° C. and kept warm for 30 min. The temperature was lowered to 31° C. and inoculated and cultured for 18 h. 200 g of betamethasone epoxide and 300 g of anhydrous ethanol were added. The conversion was carried out for about 48 h, and samples were taken. The conversion rate was 85.16% when detected by HPLC.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for improving the conversion rate of betamethasone epoxide dehydrogenation, characterized in that The following steps are involved: Step 1: Add the culture medium into the fermentation tank, and perform heating and sterilization, cooling, and inoculation in sequence to culture the bacteria; Step 2: After the cultivation is completed, betamethasone epoxide and anhydrous ethanol are added in multiple times, and the weight ratio of betamethasone epoxide to anhydrous ethanol is 10: (10-15). After the conversion rate reaches the standard, the temperature is increased and the tank is released.

2. The method for improving the conversion rate of betamethasone epoxide dehydrogenate according to claim 1, characterized in that: In the step 1, the bacterial strain used is Arthrobacter simplex, with a preservation number of CPCC 141432.

3. The method for improving the conversion rate of betamethasone epoxide dehydrogenate according to claim 1, characterized in that: The culture medium comprises, by weight, 90-110 parts of glucose, 60-80 parts of peptone, 60-80 parts of corn steep liquor, 4-6 parts of potassium dihydrogen phosphate and 10,000 parts of water.

4. The method for improving the conversion rate of betamethasone epoxide dehydrogenate according to claim 3, characterized in that: The culture medium comprises, by weight, 100 parts of glucose, 70 parts of peptone, 70 parts of corn steep liquor, 5 parts of potassium dihydrogen phosphate and 10,000 parts of water.

5. The method for improving the conversion rate of betamethasone epoxide dehydrogenate according to claim 1, characterized in that: In the step 1, the temperature is raised to 121-130° C. and kept warm for 20-30 minutes.

6. The method for improving the conversion rate of betamethasone epoxide dehydrogenate according to claim 1, characterized in that: In the step 1, the temperature is lowered to 29-31°C.

7. The method for improving the conversion rate of betamethasone epoxide dehydrogenate according to claim 1, characterized in that: In step 1, the bacteria are cultured for 12-24 hours.

8. The method for improving the conversion rate of betamethasone epoxide dehydrogenate according to claim 1, characterized in that: Betamethasone epoxy and anhydrous ethanol are administered in multiple doses with an interval of 8-12 hours between each administration.

9. The method for improving the conversion rate of betamethasone epoxide dehydrogenate according to claim 1, characterized in that: The feeding times in step 2 is 4-6 times.

10. The method for improving the conversion rate of betamethasone epoxide dehydrogenate according to claim 1, characterized in that: In step 2, the temperature is raised to 80-90° C. and kept warm for 50-60 minutes.

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