A method for preparing pravastatin sodium-based bacterial agents

Abstract

This invention belongs to the field of pharmaceutical technology and microbial inoculants, specifically relating to a method for preparing a pravastatin sodium-producing inoculant. The method described in this invention involves in-depth research on the performance of a previously screened lyophilized *Microcystis aeruginosa* inoculant. The research revealed that by fine-tuning the first sublimation drying temperature during the lyophilization process to be higher than the eutectic point of the bacterial suspension solvent, this unconventional lyophilization method unexpectedly and significantly improved the survival rate and activity of the bacteria.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and microbial inoculants, specifically relating to a method for preparing a pravastatin sodium production inoculant. Background Technology

[0002] With the increasing incidence and mortality of cardiovascular diseases, medical research has gradually confirmed that the main pathological basis of cardiovascular diseases is atherosclerosis, and hyperlipidemia is the leading cause of atherosclerosis. Therefore, the importance of lipid-lowering drugs in reducing the incidence of cardiovascular diseases has attracted attention, leading to a surge in research and development of highly effective lipid-lowering drugs.

[0003] Among the many known lipid-lowering drugs, a class of compounds originating in the early 1980s—HMG-CoA reductase inhibitors—has become the most active and rapidly developing category in cardiovascular drug research due to their high cholesterol-lowering efficiency, highly selective inhibition of cholesterol synthesis, and low toxicity. Clinically, these drugs are collectively known as statins, including lovastatin, simvastatin, pravastatin, fluvastatin, cerivastatin, and atorvastatin. Compared to other drugs in the same class, pravastatin exhibits unique tissue selectivity, selectively inhibiting cholesterol synthesis in the liver and small intestine while only weakly inhibiting cholesterol synthesis in other organs, and possesses low toxicity, making it a widely used drug in clinical practice.

[0004] Currently, pravastatin sodium is produced by microbial hydroxylation of its prodrug, mevastatin, or mevastatin. It has been reported that a variety of microorganisms can convert mevastatin to pravastatin sodium, including several genera of molds (Mortierella, WO00 / 46175), Nocardia (Norcardia, US5830695), Actinomadura (Actinomadura, WO96 / 40863), Streptomyces (Streptomyces Carbopilus EP215665, Streptomyces exfoliatus WO98 / 45410), and Micromonospora. For example, the method for obtaining pravastatin sodium by efficiently converting mevastatin based on Micropolysporum oryzae disclosed in Chinese patent CN1566328A can produce pravastatin sodium efficiently and at low cost. However, in practical applications, it has been found that due to the low spore production of Micropolysporum oryzae, the conventional method of preparing microbial freeze-dried bacterial agents will result in a low number of viable bacteria, a short preservation period, and a tendency for bacterial degradation.

[0005] Therefore, developing a pravastatin sodium production agent with a high number of live bacteria and a long preservation period is of positive significance for meeting the needs of large-scale industrial production. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a method for preparing pravastatin sodium production bacterial agent, so as to solve the problem of low number of live bacteria and easy degradation of activity in the existing technology of production bacterial agent; The second technical problem to be solved by the present invention is to provide a pravastatin sodium production inoculum with better fermentation activity.

[0007] To solve the above-mentioned technical problems, the preparation method of pravastatin sodium production bacterial agent of the present invention includes the following steps: (1) The pravastatin sodium producing strain Micropolysporum pulveratum was cultured on slant agar, and the bacterial cells were collected and a solvent was added to prepare a bacterial suspension. (2) The bacterial suspension is subjected to freezing treatment; (3) The bacterial suspension is heated to a first temperature higher than the solvent eutectic point and then subjected to a first sublimation treatment; (4) Continue to subject the bacterial suspension to a second sublimation treatment and a third sublimation treatment in sequence; (5) Continue to dry the bacterial suspension, seal and store it to obtain the final product.

[0008] Specifically, in step (1), the preservation number of the *Microsorum psyllium* is CGMCC No. 0624.

[0009] Specifically, in step (1), the solvent includes skim milk.

[0010] Specifically, in step (1), the viable count in the bacterial suspension is controlled to be greater than 10. 10 CFU / ml.

[0011] Specifically, in step (1), the slant amplification culture step includes mother slant amplification and daughter slant amplification; Specifically, the skim milk is added to the sub-slant to prepare a bacterial suspension.

[0012] Preferably, the culture medium formulations for the mother slant and daughter slant independently comprise the following components: 0.3-0.5 wt% yeast extract, 0.8-1.2 wt% malt extract, 0.3-0.5 wt% glucose, and 1.5-2.5 wt% agar powder.

[0013] Specifically, in step (2), the freezing process is carried out at a temperature of -40 to -45°C for 2-4 hours.

[0014] Specifically, in step (3), the first temperature is -20~-16℃.

[0015] Specifically, in step (3), the heating rate of the first heating step is 0.3-0.8℃ / min; Preferably, the heating rate of the first heating step is 0.5℃ / min.

[0016] Specifically, in step (3), the vacuum degree of the first sublimation step is 0.05-0.2 mbar, and the time is 8-12 hours.

[0017] Specifically, in step (4), the second sublimation step includes the step of continuing to heat the bacterial suspension to a second temperature; Preferably, the second temperature is -8 to -12°C; Preferably, the second temperature is -10°C.

[0018] Specifically, the heating rate in the second heating step is 0.3-0.8℃ / min; Preferably, the heating rate of the second heating step is 0.5℃ / min.

[0019] Specifically, the vacuum level of the second sublimation step is 0.05-0.2 mbar, and the time is 8-12 hours.

[0020] Specifically, in step (4), the third sublimation step includes the step of continuing to heat the bacterial suspension to a third temperature; Preferably, the third temperature is -5 to 5°C; Preferably, the third temperature is 0°C.

[0021] Specifically, the heating rate of the third heating step is 0.3-0.8℃ / min; Preferably, the heating rate of the third heating step is 0.5℃ / min.

[0022] Specifically, the vacuum level of the third sublimation step is 0.05-0.2 mbar, and the time is 8-12 hours.

[0023] Specifically, in step (5), the drying step includes a step of continuing to heat the bacterial suspension to a fourth temperature; Preferably, the fourth temperature is 18-25°C; Preferably, the fourth temperature is 20°C.

[0024] Specifically, the heating rate of the fourth heating step is 0.3-0.8℃ / min; Preferably, the heating rate of the fourth heating step is 0.5℃ / min.

[0025] Specifically, the vacuum degree of the drying step is 0.001-0.05 mbar, and the time is 1-3 hours.

[0026] Specifically, in step (5), the sealing step is a vacuum sealing process, preferably with the vacuum level maintained above 1 mbar for 20 minutes; the storage step is preferably stored at -20°C.

[0027] The preparation method of pravastatin sodium production bacterial agent described in this invention focuses on the performance of freeze-dried bacterial agent of Micropolysporum pulveratum, which was previously screened. In the study, it was found that by adjusting the temperature of the first sublimation drying process to be higher than the eutectic point of the bacterial suspension solvent, the survival rate and activity of the bacteria were unexpectedly greatly improved based on this unconventional freeze-drying method.

[0028] In the preparation method of the pravastatin sodium production bacterial agent described in this invention, according to the conventional freeze-drying process experience in the art, when the first sublimation temperature is higher than the eutectic point, the solid substance easily dissolves into a liquid state, absorbs heat and evaporates to dry, resulting in yellowing and shrinkage of the product, which reduces the survival rate of the bacteria. Therefore, when performing freeze-drying of the bacterial agent, the first sublimation treatment is usually carried out at a temperature lower than the solvent eutectic point. However, regarding the freeze-drying effect of the screened Micropolysporum tobira in this application, the applicant's previous experiments have shown that, referring to the eutectic point of skim milk (-26℃) and the conventional freeze-drying curve design concept, in freeze-drying methods where the first sublimation temperature is equal to or lower than the eutectic point, the survival rate of the bacterial solution after freeze-drying is less than 1%, and even by adjusting the pre-freezing rate to reduce cell damage, there is no significant improvement. Therefore, in the preparation method of pravastatin sodium production bacterial agent described in this application, for the preservation of a specifically screened Micropolysporum pulveratum, an unconventional freeze-drying method is used to perform the first sublimation treatment at a point higher than the solvent eutectic point of the bacterial suspension. Although this treatment method does not conform to conventional freeze-drying experience, it unexpectedly improves the survival rate and activity of the freeze-dried Micropolysporum pulveratum bacterial agent. Compared with the freeze-drying process experience of traditional freeze-dried bacterial agents, it has achieved unexpected technical effects.

[0029] The method for preparing pravastatin sodium production bacterial agent described in this invention features a simple and easy-to-operate freeze-drying process. The prepared freeze-dried bacterial agent has a high cell survival rate, which is beneficial for long-term preservation and effectively reduces the risk of strain degradation. Detailed Implementation

[0030] Example 1 Prepare the required mother slant and daughter slant according to the following ratios: yeast extract 0.4%, malt extract 1.0%, glucose 0.3%, and agar powder 2.0%. Incubate the preserved Micropolysporum tomentosa on the mother slant and daughter slant for 10 days respectively.

[0031] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10 For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 1 below.

[0032] Table 1 Freeze-drying procedure for Example 1

[0033] Among them, temperature control time refers to the time required for the temperature in the previous stage to change to the set temperature in this stage; maintenance time refers to the heat preservation time after the set temperature in this stage is reached.

[0034] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0035] The bacterial agent preserved in this embodiment was taken, the lyophilized tube was opened and viable bacteria were counted, and the survival rate was determined to be 26.3%.

[0036] As can be seen, the preparation method of pravastatin sodium production agent described in this embodiment greatly improves the cell survival rate by appropriately increasing the first sublimation temperature. Although this does not conform to the freeze-drying experience of conventional strains, it significantly improves the cell survival rate of the above-mentioned Micropolyspora pulverans. At the same time, it avoids the defects of traditional strains when sublimating at temperatures above the eutectic point, where solid substances easily dissolve into liquid and evaporate and dry with heat, resulting in yellowing and shrinkage of the product, which in turn reduces the cell survival rate.

[0037] Example 2 Prepare the required mother slant and daughter slant according to the following ratios: yeast extract 0.4%, malt extract 1.2%, glucose 0.4%, and agar powder 1.5%. Incubate the preserved Micropolysporum tomentosa on the mother slant and daughter slant for 10 days respectively.

[0038] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10 For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 2 below.

[0039] Table 2 Freeze-drying procedure for Example 2

[0040] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0041] The bacterial agent preserved in this embodiment was taken, the lyophilized tube was opened and viable bacteria were counted, and the survival rate was determined to be 37.4%.

[0042] Example 3 Prepare the required mother slant and daughter slant according to the following ratios: yeast extract 0.3%, malt extract 1.2%, glucose 0.5%, agar powder 1.5%. Incubate the preserved Micropolysporum tomentosa on the mother slant and daughter slant for 10 days respectively.

[0043] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 3 below.

[0044] Table 3 Freeze-drying procedure for Example 3

[0045] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0046] The bacterial agent preserved in this embodiment was taken, the lyophilized tube was opened and viable bacteria were counted, and the survival rate was determined to be 46.3%.

[0047] Example 4 Prepare the required mother slant and daughter slant according to the following ratios: yeast extract 0.5%, malt extract 1.0%, glucose 0.4%, and agar powder 2.5%. Incubate the preserved Micropolysporum tomentosa on the mother slant and daughter slant for 10 days respectively.

[0048] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10 For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 4 below.

[0049] Table 4 Freeze-drying procedure for Example 4

[0050] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0051] The bacterial agent preserved in this embodiment was taken, the lyophilized tube was opened and viable bacteria were counted, and the survival rate was determined to be 38.4%.

[0052] Example 5 Prepare the required mother slant and daughter slant according to the following ratios: yeast extract 0.5%, malt extract 1.0%, glucose 0.4%, and agar powder 2.0%. Incubate the preserved Micropolysporum tomentosa on the mother slant and daughter slant for 10 days respectively.

[0053] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10 For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 5 below.

[0054] Table 5 Freeze-drying procedure for Example 5

[0055] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0056] The bacterial agent preserved in this embodiment was taken, the lyophilized tube was opened and viable bacteria were counted, and the survival rate was determined to be 42.5%.

[0057] Example 6 Prepare the required mother slant and daughter slant according to the following ratios: yeast extract 0.5%, malt extract 1.2%, glucose 0.3%, and agar powder 2.0%. Incubate the preserved Micropolysporum tomentosa on the mother slant and daughter slant for 10 days respectively.

[0058] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10 For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 6 below.

[0059] Table 6 Freeze-drying procedure for Example 6

[0060] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0061] The bacterial agent preserved in this embodiment was taken, the lyophilized tube was opened and viable bacteria were counted, and the survival rate was determined to be 39.7%.

[0062] Comparative Example 1 Prepare the required mother slant and daughter slant according to the following ratios: yeast extract 0.3%, malt extract 1.2%, glucose 0.4%, and agar powder 2.0%. Incubate the preserved Micropolysporum tomentosa on the mother slant and daughter slant for 10 days respectively.

[0063] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10 For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 7 below.

[0064] Table 7 Freeze-drying procedure for Comparative Example 1

[0065] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0066] Take the bacterial agent preserved in the comparative sample, open the lyophilized tube and count the viable bacteria. The survival rate was determined to be 0.43%.

[0067] It is evident that this comparative scheme, based on traditional freeze-drying process experience and referencing the eutectic point of skim milk (-26℃) and conventional freeze-drying curve design, involves a first sublimation treatment at a temperature below the solvent eutectic point (-26℃ for skim milk). The survival rate of the bacterial culture after freeze-drying is less than 1%, making it unsuitable for the specific microsporidian strains selected by the process of this invention.

[0068] Comparative Example 2 Prepare the required mother slant and daughter slant according to the following ratios: yeast extract 0.4%, malt extract 1.0%, glucose 0.5%, and agar powder 2.5%. Incubate the preserved Micropolysporum tomentosa on the mother slant and daughter slant for 10 days respectively.

[0069] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10 For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 8 below.

[0070] Table 8 Freeze-drying procedure for Comparative Example 2

[0071] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0072] Take the bacterial agent preserved in the comparative sample, open the lyophilized tube and count the viable bacteria. The survival rate was determined to be 0.24%.

[0073] It is evident that this comparative scheme, based on traditional freeze-drying process experience, and referencing the eutectic point of skim milk (-26℃) and conventional freeze-drying curve design principles, sets a pre-freezing temperature of -40℃ and a first sublimation temperature of -26℃, i.e., the first sublimation treatment is performed at the solvent eutectic point temperature. It was found that the survival rate of the bacterial culture after freeze-drying was less than 1%, and simply adjusting the pre-freezing rate to reduce cell damage did not significantly improve the situation. Therefore, it is not suitable for the freeze-drying preservation of the *Microcystis aeruginosa* strain selected by the process characteristics of this invention.

[0074] Comparative Example 3 Prepare the required mother slant and daughter slant according to the following ratios: yeast extract 0.4%, malt extract 1.2%, glucose 0.4%, and agar powder 1.5%. Incubate the preserved Micropolysporum tomentosa on the mother slant and daughter slant for 10 days respectively.

[0075] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10 For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 9 below.

[0076] Table 9 Freeze-drying procedure for Comparative Example 3

[0077] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0078] The bacterial agent preserved in this embodiment was taken, and the lyophilization tube was opened for viable cell counting. The survival rate was measured to be 21.5%. It can be seen that setting the temperature of the first stage of sublimation to -15°C still resulted in a high survival rate of the bacterial strain. However, due to the excessively high temperature in this stage, local melting occurred, resulting in poor morphology of the lyophilized powder. If a higher temperature is set in this stage, the entire bacterial solution will melt and become impossible to freeze-dry.

[0079] Comparative Example 4 The required mother slant and daughter slant were prepared according to the following ratios: glucose 3%, yeast extract 2%, soybean peptone 0.5%, K2HPO4·3H2O 0.2%, and agar powder 1.5%. The preserved Madura actinomycetes were cultured on the mother slant and daughter slant for 10 days respectively.

[0080] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10 For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 10 below.

[0081] Table 10 Freeze-drying procedure for Comparative Example 4

[0082] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0083] The bacterial agent preserved in this embodiment was taken, the lyophilized tube was opened and viable bacteria were counted, and the survival rate was determined to be 28.6%.

[0084] It is evident that, for conventional pravastatin sodium fermentation strains, the traditional approach of designing freeze-drying programs based on the solvent eutectic point (-26℃) and conventional freeze-drying curves can fully meet the survival rate requirements of conventional pravastatin sodium fermentation strains.

[0085] Comparative Example 5 The required mother slant and daughter slant were prepared according to the following ratios: glucose 3%, yeast extract 2%, soybean peptone 0.5%, K2HPO4·3H2O 0.2%, and agar powder 1.5%. The preserved Madura actinomycetes were cultured on the mother slant and daughter slant for 10 days respectively.

[0086] Skim milk was added to the sub-slant and mixed to prepare a uniform bacterial suspension, with the number of viable bacteria in the suspension controlled to reach 10. 10 For concentrations of CFU / ml or higher, dispense the bacterial solution into sterile ampoules and freeze-dry according to the procedure in Table 11 below.

[0087] Table 11 Freeze-drying procedure for Comparative Example 5

[0088] After freeze-drying, connect it to the measuring tube of the manifold, use a vacuum pump to evacuate, maintain the vacuum level above 1 mbar for 20 minutes, and then ignite and seal it using an ampoule sealing machine.

[0089] The bacterial agent preserved in this embodiment was taken, the lyophilized tube was opened and viable bacteria were counted, and the survival rate was determined to be 25.4%.

[0090] It is evident that for conventional pravastatin sodium fermentation strains, the traditional approach of designing freeze-drying programs based on the solvent eutectic point (-26℃) and conventional freeze-drying curves results in a higher survival rate of the freeze-dried bacterial agent compared to the sublimation treatment at temperatures above the eutectic point.

[0091] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a pravastatin sodium-based microbial agent, characterized in that, Includes the following steps: (1) Take the pravastatin sodium production strain Micropolysporum pulveratum ( Micropolyspora roseoalba) The bacteria were amplified by slant culture, and the bacterial cells were collected and a solvent was added to prepare a bacterial suspension; the solvent was skim milk; the preservation number of the *Microcystis aeruginosa* was CGMCC No. 0624. (2) The bacterial suspension is subjected to freezing treatment; (3) The bacterial suspension is heated to a first temperature higher than the solvent eutectic point and then subjected to a first sublimation treatment; the heating rate of the first heating step is 0.3-0.8℃ / min; the vacuum degree of the first sublimation step is 0.05-0.2mbar and the time is 8-12 hours; the first temperature is -20~-16℃. (4) Continue to subject the bacterial suspension to a second sublimation treatment and a third sublimation treatment in sequence; (5) Continue to dry the bacterial suspension, seal and store it to obtain the final product.

2. The method for preparing the pravastatin sodium production agent according to claim 1, characterized in that, In step (1), the number of viable bacteria in the bacterial suspension is controlled to be greater than 10. 10 CFU / ml.

3. The method for preparing the pravastatin sodium production agent according to any one of claims 1-2, characterized in that, In step (1), the slant amplification culture step includes mother slant amplification and daughter slant amplification.

4. The method for preparing the pravastatin sodium production agent according to claim 3, characterized in that, In step (1), the culture medium formulations of the mother slant and daughter slant independently include the following components: yeast extract 0.3-0.5wt%, malt extract 0.8-1.2wt%, glucose 0.3-0.5wt%, and agar powder 1.5-2.5wt%.

5. The method for preparing pravastatin sodium production microbial agent according to any one of claims 1-2 and 4, characterized in that, In step (2), the freezing process is carried out at a temperature of -45 to -40°C for 2 to 4 hours.

6. The method for preparing pravastatin sodium production microbial agent according to any one of claims 1-2 and 4, characterized in that, In step (3), the heating rate of the first heating step is 0.5℃ / min.

7. The method for preparing pravastatin sodium production microbial agent according to any one of claims 1-2 and 4, characterized in that, In step (4), the second sublimation step includes the step of continuing to heat the bacterial suspension to a second temperature.

8. The method for preparing the pravastatin sodium production agent according to claim 7, characterized in that, The second temperature is -12 to -8℃.

9. The method for preparing the pravastatin sodium production agent according to claim 7, characterized in that, The second temperature is -10℃.

10. The method for preparing the pravastatin sodium production agent according to claim 7, characterized in that, The heating rate of the second heating step is 0.3-0.8℃ / min.

11. The method for preparing the pravastatin sodium production agent according to claim 7, characterized in that, The heating rate in the second heating step is 0.5℃ / min.

12. The method for preparing pravastatin sodium production bacterial agent according to any one of claims 1-2, 4, 8-11, characterized in that, The vacuum level for the second sublimation step is 0.05-0.2 mbar, and the time is 8-12 hours.

13. The method for preparing pravastatin sodium production bacterial agent according to any one of claims 1-2, 4, 8-11, characterized in that, In step (4), the third sublimation step includes the step of continuing to heat the bacterial suspension to a third temperature.

14. The method for preparing the pravastatin sodium production agent according to claim 13, characterized in that, The third temperature is -5~5℃.

15. The method for preparing the pravastatin sodium production agent according to claim 13, characterized in that, The third temperature is 0°C.

16. The method for preparing the pravastatin sodium production agent according to claim 13, characterized in that, The heating rate of the third heating step is 0.3-0.8℃ / min.

17. The method for preparing the pravastatin sodium production agent according to claim 13, characterized in that, The heating rate of the third heating step is 0.5℃ / min.

18. The method for preparing pravastatin sodium production microbial agent according to any one of claims 1-2, 4, 8-11, 14-17, characterized in that, The vacuum level of the third sublimation step is 0.05-0.2 mbar, and the time is 8-12 hours.

19. The method for preparing pravastatin sodium production bacterial agent according to any one of claims 1-2, 4, 8-11, 14-17, characterized in that, In step (5), the drying step includes a step of continuing to heat the bacterial suspension to a fourth temperature.

20. The method for preparing the pravastatin sodium production agent according to claim 19, characterized in that, The fourth temperature is 18-25℃.

21. The method for preparing the pravastatin sodium production agent according to claim 19, characterized in that, The fourth temperature is 20°C.

22. The method for preparing the pravastatin sodium production agent according to claim 19, characterized in that, The heating rate of the fourth heating step is 0.3-0.8℃ / min.

23. The method for preparing the pravastatin sodium production agent according to claim 19, characterized in that, The heating rate of the fourth heating step is 0.5℃ / min.

24. The method for preparing pravastatin sodium production microbial agent according to any one of claims 1-2, 4, 8-11, 14-17, and 20-23, characterized in that, The drying step is performed under a vacuum of 0.005-0.05 mbar for 1-3 hours.

Images