Process for the extraction of fusidic acid hemihydrate from fusidic acid bacterial sludge

Abstract

The application provides a method for extracting fusidic acid hemihydrate from fusidic acid bacterial residue, belongs to the field of fermentation engineering, and reutilizes the bacterial residue after separation of an extraction liquid, breaks through the limitation of separation and purification of the extraction liquid in the prior art, and significantly improves the potency, yield, energy saving and emission reduction and pollution reduction of the preparation of fusidic acid.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fermentation engineering, in particular to a method for extracting fusidic acid hemihydrate from fusidic acid bacterial residue. BACKGROUND

[0002] Fusidic acid is a kind of fusidic acid antibiotic with a steroid skeleton produced by the fungus Cephalosporium globosum or some cephalosporins. Its pharmacological mechanism is mainly to inhibit the synthesis of bacterial proteins to play an antibacterial role, especially for staphylococcus (including methicillin-resistant strains or other antibiotic-resistant strains), which has strong antibacterial effect, and also has certain antibacterial effect on streptococcus, enterococcus, diphtheria bacillus, clostridium, neisseria and tubercle bacillus.

[0003] Fusidic acid is mainly prepared by fermentation method. The existing technology extracts and separates fusidic acid from the fermentation broth, including extraction method, resin method, etc. For example, CN101812498A discloses a fermentation production method of fusidic acid, which improves the titer of fusidic acid by culturing seeds, inoculation, and batch feeding of culture medium fermentation. CN101792476A obtains a crystal body by filtering and centrifuging the fusidic acid fermentation broth, and then concentrating and extracting.

[0004] The existing technology lacks a process for directly extracting and purifying fusidic acid from bacterial residue. After fermentation, about 5-15% of fusidic acid is left in the bacterial residue. Even if the extraction liquid is separated and concentrated, there is a lot of solvent loss and pollution, and the bacterial residue is completely wasted, etc. Therefore, the process needs to be optimized. At the same time, due to the cross influence of various biological and chemical factors in the fermentation process, any change in the extraction process elements will affect the components, thereby affecting the yield and purity. For microbial fermentation products, how to effectively improve the yield is the main problem faced by industrial manufacturers. SUMMARY

[0005] (I) Technical problems solved

[0006] In view of the technical problems in the extraction of fusidic acid fermentation production on the market, especially the problems of large solvent dosage and high cost, the present application reuses the bacterial residue after separation of the extraction liquid, breaks through the limitations of the existing technology for separation and purification of the extraction liquid, and significantly improves the preparation of fusidic acid in terms of titer, yield, energy saving and emission reduction, and pollution reduction.

[0007] (II) Technical solutions

[0008] In order to achieve the above purposes, the present application is realized by the following technical solutions:

[0009] The present application claims a method for extracting fusidic acid hemihydrate from fusidic acid bacterial residue, characterized in that the steps are as follows:

[0010] 1) Add fusidic acid residue to a pressure vessel, add organic solvent, pressurize and fill with nitrogen, stir and then release pressure;

[0011] 2) After releasing pressure, take a sample of the mixed solution, centrifuge and then take the supernatant to test the titer;

[0012] 3) Filter the soaking solution in the pressure vessel, decolorize and recrystallize to obtain fusidic acid hemihydrate.

[0013] Preferably, the organic solvent is ethyl acetate, and the pressure in the pressure vessel is ≥0.5 Mpa,

[0014] Further, the pressure in the pressure vessel is ≥1.5 Mpa, preferably 1.5-3 Mpa.

[0015] Further, the reaction temperature is controlled at 20-25℃, and the stirring time is ≥2 h.

[0016] Further, the stirring time is more preferably ≥6 h, and most preferably 6-12 h.

[0017] Further, the ratio of the mass (kg) of the residue to the volume (L) of the organic solvent is 1:(2-5) kg / L.

[0018] Further, the ratio of the mass (kg) of the residue to the volume (L) of the organic solvent is 1:3 kg / L or 1:5 kg / L.

[0019] Further, the titer is ≥6500 mg / L, preferably ≥6600 mg / L, more preferably ≥6700 mg / L, and more preferably ≥6800 mg / L.

[0020] In another technical solution, the titer is ≥10000 mg / L.

[0021] The present application also includes a method for extracting fusidic acid, which comprises the above steps of extracting fusidic acid hemihydrate from fusidic acid residue.

[0022] All raw materials and reagents in the specification of the present application are commercially available or prepared by conventional experimental methods.

[0023] (Three) beneficial effects

[0024] The present application provides a method for extracting fusidic acid hemihydrate from fusidic acid residue. Compared with the prior art, the present application has the following beneficial effects:

[0025] 1. The product obtained by the present application has high separation purity and high titer, which is beneficial for storage and batch modular production.

[0026] 2. By simplifying the process, the limitations of the prior art for the separation and purification of the extract are broken through; the extraction of the fungus residue obtains considerable yield, greatly reduces the cost, and reduces environmental pollution. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0028] Figure 1 LCMS spectrum 1 of the crystalline product of fusidic acid hemihydrate of Example 1;

[0029] Figure 2 LCMS spectrum 2 of the crystalline product of fusidic acid hemihydrate of Example 1.

[0030] Figure 3 Hydrogen spectrum of fusidic acid.

[0031] Figure 4 Carbon spectrum of fusidic acid.

[0032] Figure 5 Infrared spectrum of fusidic acid.

[0033] Figure 6 Liquid chromatogram of the crystalline product of fusidic acid hemihydrate. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application are described clearly and completely. Obviously, the described embodiments are part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0035] In order to better understand the above technical solutions, the above technical solutions will be described in detail in combination with the drawings in the specification and the specific embodiments.

[0036] The fusidic acid bacterial residue in the embodiment of the present application is the residual bacterial residue after the separation or pressing of the fusidic acid fermentation liquor in the prior art. The fermentation liquor preparation method can refer to the preparation of the fusidic acid fermentation liquor in CN101812498A, which is fully incorporated herein, but does not limit the protection scope of the present application in any way, and only illustrates the conventional fermentation method. The fusidic acid bacterial residue described in the present application is the residual bacterial residue after the separation of the fermentation liquor or the concentrated liquor of the fermentation liquor prepared by the conventional method.

[0037] Embodiment 1:

[0038] The method for extracting fusidic acid hemihydrate from the fusidic acid bacterial residue specifically comprises the following steps:

[0039] 1 kg of fusidic acid bacterial residue is added into a 10 L pressure tank, 5 L of ethyl acetate is added, the nitrogen gas is pressurized to 0.5 MPa, the temperature is controlled at 20-25°C, and the stirring is performed for 6 h. After the pressure is released, 5 ml of the bacterial residue-ethyl acetate mixed liquid sample is taken, the supernatant is taken after centrifugation, and the titer is detected to be 6863 mg / L. After the soaking liquid in the pressure tank is filtered, the fusidic acid hemihydrate qualified product fine powder 30.1 g is obtained after the steps of decolorization and recrystallization.

[0040] Embodiment 2:

[0041] 1 kg of the same batch of bacterial residue is added into a 10 L pressure tank, 5 L of ethyl acetate is added, the nitrogen gas is pressurized to 0.5 MPa, the temperature is controlled at 20-25°C, and the stirring is performed for 12 h. After the pressure is released, 5 ml of the bacterial residue-ethyl acetate mixed liquid sample is taken, the supernatant is taken after centrifugation, and the titer is detected to be 6870 mg / L. After the soaking liquid in the pressure tank is filtered, the fusidic acid hemihydrate qualified product fine powder 30.3 g is obtained after the steps of decolorization and recrystallization.

[0042] Embodiment 3:

[0043] 1 kg of the same batch of bacterial residue is added into a 10 L pressure tank, 5 L of ethyl acetate is added, the nitrogen gas is pressurized to 0.5 MPa, the temperature is controlled at 20-25°C, and the stirring is performed for 18 h. After the pressure is released, 5 ml of the mixed liquid sample is taken, the supernatant is taken after centrifugation, and the titer is detected to be 6882 mg / L. After the soaking liquid in the pressure tank is filtered, the fusidic acid hemihydrate qualified product fine powder 30.5 g is obtained after the steps of decolorization and recrystallization.

[0044] The titer of the soaking liquid under different stirring times is as follows:

[0045]

[0046] Embodiment 4, the difference from embodiment 1 is:

[0047] The nitrogen gas is pressurized to 1 MPa.

[0048] Embodiment 5, the difference from embodiment 1 is:

[0049] Nitrogen pressure 1.5 MPa.

[0050] Example 6, which is different from example 1:

[0051] Nitrogen pressure 3 MPa.

[0052] Under different pressures, 20-25℃ stirring soak for 6h, the titer of the soak solution is as follows:

[0053]

[0054] The above products are detected: all are fusidic acid hemihydrate qualified products, purity 99.45-99.67% (detection spectrum see Figures 1-5 ).

[0055] Example 7:

[0056] The method for extracting fusidic acid hemihydrate from fusidic acid bacterial residue comprises the following steps:

[0057] 1kg of fusidic acid bacterial residue is added to a 10L pressure kettle, 3L of ethyl acetate is added, nitrogen pressure is 0.5MPa, temperature is controlled at 20-25℃, stirring for 6h, after pressure relief, 5ml of mixed solution sample is taken, after centrifugation, the titer of the supernatant is 11503mg / L.

[0058] After the soak solution is filtered, the filter cake bacterial residue is added to a 10L pressure kettle, 3L of ethyl acetate is added, nitrogen pressure is 0.5MPa, temperature is controlled at 20-25℃, stirring for 6h, secondary soaking is carried out, after pressure relief, 2ml of mixed solution sample is taken, after centrifugation, the titer of the supernatant is 23mg / L.

[0059] After the secondary soak solution is filtered, the filter cake bacterial residue is added to a 10L pressure kettle, 3L of ethyl acetate is added, nitrogen pressure is 0.5MPa, temperature is controlled at 20-25℃, stirring for 6h, tertiary soaking is carried out, after pressure relief, 2ml of mixed solution sample is taken, after centrifugation, the titer of the supernatant is 16mg / L.

[0060] In the above examples, through the titer detection after multiple soakings of the bacterial residue, it is known that the titer gradually decreases with the increase of the soaking times, considering the cost and efficiency in the actual process, the soaking extraction method of the present scheme is preferred, and the one-time soaking method can be used.

[0061] Comparative example 1:

[0062] The method for extracting fusidic acid hemihydrate from fusidic acid bacterial residue comprises the following steps:

[0063] 10L pressure kettle is added with 1 kg fusidic acid bacterial residue, methanol 5L, nitrogen pressure 0.5 MPa, temperature control 20-25℃, stirring for 6h, after pressure relief, 5ml of mixed liquid sample is taken, after centrifugation, the supernatant is detected for a titer of 6586mg / L, the soaking liquid in the pressure kettle is filtered, and after decolorization, recrystallization and other steps, 29g of fusidic acid hemihydrate fine powder of qualified product is obtained, with a purity of 99% (detection spectrum is the same as that in Example 1).

[0064] Comparative Example 2:

[0065] 10L three-necked flask is added with 1 kg of fusidic acid bacterial residue, ethyl acetate 5L, temperature control 20-25℃, stirring for 6h, 5ml of mixed liquid sample is taken, after centrifugation, the supernatant is detected for a titer of 6587mg / L, the soaking liquid in the pressure kettle is filtered, and after decolorization, recrystallization and other steps, 28.9g of fusidic acid hemihydrate fine powder of qualified product is obtained, with a purity of 98.78%.

[0066] Comparative Example 3, the difference between Comparative Example 2 and Comparative Example 3 is:

[0067] The soaking time of the same batch of bacterial residue is extended to 12h, and the soaking liquid is detected for a titer of 6593mg / L.

[0068] Comparative Example 4, the difference between Comparative Example 2 and Comparative Example 4 is:

[0069] The soaking time of the same batch of bacterial residue is extended to 18h, and the soaking liquid is detected for a titer of 6603mg / L.

[0070] Comparative Example 5:

[0071] 10L three-necked flask is added with 1 kg of fusidic acid bacterial residue, methanol 5L, temperature control 20-25℃, stirring for 6h, 5ml of mixed liquid sample is taken, after centrifugation, the supernatant is detected for a titer of 6375mg / L, the soaking liquid in the pressure kettle is filtered, and after decolorization, recrystallization and other steps, 28g of fusidic acid hemihydrate fine powder of qualified product is obtained, with a purity of 98.67%.

[0072] The qualified fusidic acid product obtained by recrystallization in the above examples can be realized by the following operation:

[0073] After the fusidic acid bacterial residue is soaked with ethyl acetate, filtered, washed and decolorized, part of the ethyl acetate is concentrated until the titer of the sample is 50000mg / L, the temperature is lowered to room temperature, methanol is added to dilute to a titer of 30000mg / L, stirred for 20min, transferred to an environment of 0-10℃ and left overnight, centrifuged and washed with a mixture of petroleum ether and methanol (petroleum ether:methanol=1:1) to obtain a crude product.

[0074] Crystallization: the dried crude product is mixed with ethanol in a 1:1 mass ratio and added to the reaction bottle, stirred to dissolve, the temperature is controlled not to exceed 50 DEG C, the mixed solution of crude product and methanol (crude product:methanol = 1:1) is added dropwise, the dropwise adding time is controlled within 1h, after the dropwise adding is completed, continue to stir to dissolve, after the dissolution is basically complete, the mixed solution of crude product and water (crude product:water = 1:0.5) is added dropwise, the dropwise adding speed is controlled to be completed within 1h, after the dropwise adding is completed, slowly cool to 6±0.5 DEG C, low speed stirring for 2h, centrifugation to obtain fusidic acid hemihydrate, the product HPLC is detected as shown in the attached Figure 6

[0075] The above test results show that, through the preparation process of the bacterial residue, a product with high purity is obtained, it can be seen that the selection of the solvent and the pressure of the autoclave are highly related to the purity of the product, but in a specific case, increasing the soaking time and increasing the pressure cannot increase the titer and purity, which can prove that due to the cross influence of various biological and chemical factors in the fermentation process, the unpredictability of the influence of the slight process change of the bacterial residue extraction on the result, and the reuse of the bacterial residue will not increase the titer by secondary extraction (soaking). On this basis, the present application can completely obtain a more simplified and optimized process.

[0076] In summary, compared with the prior art, the present application has the following beneficial effects:

[0077] 1. The product obtained by the present application has high separation purity and high titer, which is beneficial to storage and batch modular production.

[0078] 2. By simplifying the process, the limitations of the prior art for separating and purifying the extraction liquid are broken through; the bacterial residue extraction obtains a considerable yield, greatly reduces the cost, and reduces environmental pollution.

[0079] It should be noted that, in this text, relational terms such as first and second are used merely to distinguish one entity or action from another, and do not necessarily require or imply that there is any such actual relationship or order between these entities or actions. Moreover, the terms "include", "contain" or any other variant thereof are intended to cover non-exclusive inclusion, so that the process, method, article or equipment including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or equipment. Without more limitations, the element defined by the statement "including a" does not exclude the presence of another identical element in the process, method, article or equipment including the element.

[0080] ​The above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can be modified, or some technical features can be replaced by equivalent features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for extracting fusidic acid hemihydrate from fusidic acid bacteria residue, characterized in that, The steps are as follows: S1. Add the fusidic acid bacteria residue to the pressure vessel, add organic solvent, pressurize and purge with inert gas, stir for ≥2h and then release the pressure; Wherein, the organic solvent is ethyl acetate, and the pressure vessel pressure is ≥0.5 MPa; S2. After depressurization, take a sample of the mixture of bacterial residue and organic solvent, centrifuge it, and then take the supernatant after settling to test the potency. S3. The bacterial residue soaking solution in the pressure vessel is filtered, decolorized, and recrystallized to obtain fusidic acid hemihydrate.

2. The method for extracting fusidic acid hemihydrate from fusidic acid bacteria residue as described in claim 1, characterized in that, The temperature during the preparation process is controlled at 20-25℃, and the stirring time in step S1 is ≥6h.

3. The method for extracting fusidic acid hemihydrate from fusidic acid bacteria residue as described in claim 1, characterized in that, The mass ratio of the bacterial residue to the volume of the organic solvent is 1:(2-5) kg / L, and the detection potency is ≥6500mg / L.

4. The method for extracting fusidic acid hemihydrate from fusidic acid bacteria residue as described in claim 3, characterized in that, The mass ratio of the bacterial residue to the volume ratio of the organic solvent is 1:5 kg / L.

5. The method for extracting fusidic acid hemihydrate from fusidic acid bacteria residue as described in claim 3, characterized in that, The mass ratio of the bacterial residue to the volume ratio of the organic solvent is 1:3 kg / L.

6. The method for extracting fusidic acid hemihydrate from fusidic acid bacteria residue as described in claim 5, characterized in that, The detection potency is ≥10000mg / L.

7. A method for extracting fusidic acid, characterized in that, Includes the preparation steps contained in the method for extracting fusidic acid hemihydrate from fusidic acid bacteria residue as described in any one of claims 1-6.

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