Method for extracting fusidic acid hemihydrate from fusidic acid bacterial residues

Abstract

The invention provides a method for extracting fusidic acid hemihydrate from fusidic acid fungus residues, belongs to the field of fermentation engineering, aims at reutilizing fungus residues obtained after separation of an extracting solution, breaks through the limitation of separation and purification of the extracting solution in the prior art, and improves the yield of fusidic acid. The preparation of fusidic acid is remarkably improved in the aspects of titer, yield, energy conservation, emission reduction and pollution reduction.

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 steroidal 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 against staphylococcus (including methicillin-resistant strains or other antibiotic-resistant strains), and also has certain antibacterial effect on streptococcus, enterococcus, diphtheria bacillus, clostridium, neisseria and mycobacterium tuberculosis.

[0003] Fusidic acid is mainly prepared by fermentation. The prior art extracts, separates and concentrates fusidic acid from fermentation broth, including extraction, resin method, etc. For example, CN101812498A discloses a fermentation production method of fusidic acid, which improves the titer of fusidic acid by culturing seeds, inoculating, and supplementing culture medium in batches. CN101792476A obtains a crystal body by filtering and centrifuging the fusidic acid fermentation broth, concentrating and extracting, etc.

[0004] The prior art lacks a process for directly extracting and purifying fusidic acid from bacterial residue. After fermentation, about 5-15% of fusidic acid remains 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 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 prior art 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.

[0006] (II) Technical solutions In order to achieve the above purpose, the present application is realized by the following technical solutions: The present application claims a method for extracting fusidic acid hemihydrate from fusidic acid bacterial residue, characterized in that the steps are as follows: 1) Add fusidic acid bacterial residue into a pressure vessel, add organic solvent, pressurize and fill nitrogen, stir and then release pressure; 2) After releasing pressure, take the mixed liquid sample, centrifuge and then take the supernatant to detect the titer; 3) The soaking liquid in the pressure vessel is filtered, decolorized and recrystallized to obtain fusidic acid hemihydrate.

[0007] Preferably, the organic solvent is ethyl acetate, and the pressure in the pressure vessel is ≥0.5Mpa, Further, the pressure in the pressure vessel is ≥1.5Mpa, preferably 1.5-3Mpa.

[0008] Further, the reaction temperature is controlled at 20-25℃, and the stirring time is ≥2h.

[0009] Further, the stirring time is more preferably ≥6h, and most preferably 6-12h.

[0010] Further, the mass (kg) of the bacterial residue and the volume (L) of the organic solvent are in a ratio of 1:(2-5) kg / L.

[0011] Further, the mass (kg) of the bacterial residue and the volume (L) of the organic solvent are in a ratio of 1:3 kg / L or 1:5 kg / L.

[0012] Further, the detection titer is ≥6500mg / L, preferably ≥6600mg / L, more preferably ≥6700mg / L, and more preferably ≥6800mg / L.

[0013] In another technical solution, the detection titer is ≥10000mg / L.

[0014] The present application also includes a method for extracting fusidic acid, which comprises the above steps of extracting fusidic acid hemihydrate from fusidic acid bacterial residue.

[0015] All raw materials and reagents in the specification of the present application are commercially available or prepared by conventional experimental methods.

[0016] (Three) beneficial effects The present application provides a method for extracting fusidic acid hemihydrate from fusidic acid bacterial residue. Compared with the prior art, the following beneficial effects are achieved: 1. The product obtained by the present application has high separation purity and high titer, which is beneficial for storage and batch modular production.

[0017] 2. By simplifying the process, the limitations of the prior art for extracting liquid separation and purification are broken through; the extraction of bacterial residue obtains considerable yield, greatly reduces the cost, and reduces environmental pollution. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can be obtained without creative labor on the basis of these drawings.

[0019] Figure 1 LCMS chart 1 of the crystalline product of fusidic acid hemihydrate in Example 1; Figure 2 LCMS chart 2 of the crystalline product of fusidic acid hemihydrate in Example 1.

[0020] Figure 3 NMR chart of fusidic acid.

[0021] Figure 4 Carbon spectrum of fusidic acid.

[0022] Figure 5 Infrared chart of fusidic acid.

[0023] Figure 6 Liquid chromatogram of the crystalline product of fusidic acid hemihydrate. DETAILED DESCRIPTION

[0024] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are some of the embodiments of the present application, but 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 fall within the protection scope of the present application.

[0025] In order to better understand the above technical solutions, the above technical solutions will be described in detail below in combination with the accompanying drawings of the specification and specific embodiments.

[0026] The fusidic acid residue in the embodiments of the present application is the residual residue after the separation or pressing of the fusidic acid fermentation broth in the prior art. The preparation method of the fermentation broth can refer to the preparation of the fusidic acid fermentation broth in CN101812498A, which is fully introduced 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 residue described in the present application is the residual residue after the separation of the fermentation broth or the concentrated liquid of the fermentation broth prepared by the conventional method.

[0027] Example 1: The method for extracting fusidic acid hemihydrate from fusidic acid residue specifically comprises the following steps: 10L pressure kettle is added with 1 kg of fusidic acid bacteria residue, 5 L of ethyl acetate, nitrogen is filled to 0.5 MPa, stirring is controlled at 20-25 DEG C for 6 h, after pressure relief, 5 ml of the mixture of the bacteria residue and ethyl acetate is taken, after centrifugation, the supernatant is taken for detection, the titer is 6863 mg / L, the soaking liquid in the pressure kettle is filtered, after decolorization, recrystallization and other steps, 30.1 g of qualified fusidic acid hemihydrate fine powder is obtained.

[0028] Example 2: 1 kg of the same batch of bacteria residue is added to a 10 L pressure kettle, 5 L of ethyl acetate is added, nitrogen is filled to 0.5 MPa, stirring is controlled at 20-25 DEG C for 12 h, after pressure relief, 5 ml of the mixture is taken, after centrifugation, the supernatant is taken for detection, the titer is 6870 mg / L, the soaking liquid in the pressure kettle is filtered, after decolorization, recrystallization and other steps, 30.3 g of qualified fusidic acid hemihydrate fine powder is obtained.

[0029] Example 3: 1 kg of the same batch of bacteria residue is added to a 10 L pressure kettle, 5 L of ethyl acetate is added, nitrogen is filled to 0.5 MPa, stirring is controlled at 20-25 DEG C for 18 h, after pressure relief, 5 ml of the mixture is taken, after centrifugation, the supernatant is taken for detection, the titer is 6882 mg / L, the soaking liquid in the pressure kettle is filtered, after decolorization, recrystallization and other steps, 30.5 g of qualified fusidic acid hemihydrate fine powder is obtained.

[0030] Under different stirring times, the titer of the soaking liquid is as follows: Example 4, the difference between example 1 and example 1 is: Nitrogen is filled to 1 MPa.

[0031] Example 5, the difference between example 1 and example 1 is: Nitrogen is filled to 1.5 MPa.

[0032] Example 6, the difference between example 1 and example 1 is: Nitrogen is filled to 3 MPa.

[0033] Under different pressures, stirring is controlled at 20-25 DEG C for 6 h, the titer of the soaking liquid is as follows: The above products are detected: all are qualified fusidic acid hemihydrate products, the purity is 99.45-99.67% (the detection spectrum is shown in Figures 1-5 ).

[0034] Example 7: The method for extracting fusidic acid hemihydrate from fusidic acid bacteria residue specifically comprises the following steps: 10L pressure kettle is added with 1 kg of fusidic acid bacterial residue, 3 L of ethyl acetate, nitrogen is filled to 0.5 MPa, temperature is controlled at 20-25 DEG C, and stirring is performed for 6 h; after pressure relief, 5 ml of mixed liquid sample is taken, centrifugation is performed, and the titer of the supernatant is 11503 mg / L.

[0035] After filtration of the soaking liquid, the filter cake bacterial residue is added into a 10L pressure kettle, 3 L of ethyl acetate is added, nitrogen is filled to 0.5 MPa, temperature is controlled at 20-25 DEG C, and stirring is performed for 6 h to perform secondary soaking; after pressure relief, 2 ml of mixed liquid sample is taken, centrifugation is performed, and the titer of the supernatant is 23 mg / L.

[0036] After filtration of the secondary soaking liquid, the filter cake bacterial residue is added into a 10L pressure kettle, 3 L of ethyl acetate is added, nitrogen is filled to 0.5 MPa, temperature is controlled at 20-25 DEG C, and stirring is performed for 6 h to perform tertiary soaking; after pressure relief, 2 ml of mixed liquid sample is taken, centrifugation is performed, and the titer of the supernatant is 16 mg / L.

[0037] In the above examples, through titer detection after multiple times of soaking 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 mode of the present scheme is preferentially selected, and the soaking mode once can be adopted.

[0038] Comparative Example 1 The method for extracting fusidic acid hemihydrate from fusidic acid bacterial residue specifically comprises the following steps: 10L pressure kettle is added with 1 kg of fusidic acid bacterial residue, 5 L of methanol, nitrogen is filled to 0.5 MPa, temperature is controlled at 20-25 DEG C, and stirring is performed for 6 h; after pressure relief, 5 ml of mixed liquid sample is taken, centrifugation is performed, and the titer of the supernatant is 6586 mg / L; after filtration of the soaking liquid in the pressure kettle, fusidic acid hemihydrate qualified product fine powder 29 g with a purity of 99% is obtained through steps such as decolorization and recrystallization (the detection spectrum is the same as that in Example 1).

[0039] Comparative Example 2 10L three-necked flask is added with 1 kg of fusidic acid bacterial residue, 5 L of ethyl acetate, temperature is controlled at 20-25 DEG C, and stirring is performed for 6 h; 5 ml of mixed liquid sample is taken, centrifugation is performed, and the titer of the supernatant is 6587 mg / L; after filtration of the soaking liquid in the pressure kettle, fusidic acid hemihydrate qualified product fine powder 28.9 g with a purity of 98.78% is obtained through steps such as decolorization and recrystallization.

[0040] Comparative Example 3, which is different from Comparative Example 2 in that: The soaking time of the same batch of bacterial residue is extended to 12 h, and the titer of the soaking liquid is 6593 mg / L.

[0041] Comparative Example 4, which is different from Comparative Example 2 in that: The soaking time of the same batch of bacterial residue is extended to 18 h, and the titer of the soaking liquid is 6603 mg / L.

[0042] Comparative Example 5: In a 10L three-necked flask, 1kg of fusidic acid bacterial residue was added, 5L of methanol was added, and stirring was carried out at a temperature of 20-25℃ for 6h. 5ml of the mixed solution was taken, centrifuged, and the supernatant was detected to have a titer of 6375mg / L. After filtering the soaking solution in a pressure tank, the fusidic acid hemihydrate qualified product powder 28g was obtained after the steps of decolorization and recrystallization, and the purity was 98.67%.

[0043] The fusidic acid qualified product obtained by recrystallization in the above examples can be realized by the following operation: After the fusidic acid bacterial residue was soaked with ethyl acetate, filtered, washed, and decolorized, part of the ethyl acetate was concentrated until the titer was 50000mg / L, and then methanol was added to dilute the titer to 30000mg / L. After stirring for 20min, the solution was transferred to an environment of 0-10℃ and left overnight. After centrifugation, the crude product was obtained by elution with a mixture of petroleum ether and methanol (petroleum ether:methanol=1:1).

[0044] Crystallization: The dried crude product was mixed with ethanol at a mass ratio of 1:1 and then added to a reaction bottle. After stirring and dissolving, the temperature was controlled to be not higher than 50℃. The mixture of the crude product and methanol (crude product:methanol=1:1) was added dropwise, and the dropwise addition time was controlled to be within 1h. After the dropwise addition was completed, the stirring was continued to dissolve. After the solution was basically completely dissolved, the mixture of the crude product and water (crude product:water=1:0.5) was added dropwise after filtration. The dropwise addition was completed within 1h. After the dropwise addition was completed, the temperature was slowly reduced to 6±0.5℃, and the stirring was continued at a low speed for 2h. The fusidic acid hemihydrate was obtained by centrifugation. The product HPLC detection was as shown in the attached Figure 6 figure (if the detection is unqualified, the recrystallization operation can be repeated twice).

[0045] 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 pressure tank are highly related to the titer and purity of the product. However, in specific cases, increasing the soaking time and increasing the pressure cannot increase the titer and purity. It can be proved 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 changes of the bacterial residue extraction on the results, 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.

[0046] In summary, compared with the prior art, the present application has the following beneficial effects: 1. The product obtained by the present application has high separation purity and high titer, which is beneficial to storage and batch modular production.

[0047] 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.

[0048] It should be noted that, in this text, relational terms such as first and second are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between such entities or operations. Moreover, the terms "comprises", "comprising", or any other variation thereof are intended to cover non-exclusive inclusions, so that a process, method, article, or device that includes a series of elements not only includes those elements, but also includes other elements not explicitly listed, or inherent to such a process, method, article, or device. Without more limitations, the element defined by the statement "comprises a" does not exclude the presence of other identical elements in the process, method, article, or device that includes the element.

[0049] The above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for part of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solution 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: 1) Add the fusidic acid bacteria residue to a pressure vessel, add organic solvent, pressurize and purge with inert gas, stir for ≥2 hours, and then release the pressure; 2) 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; 3) The bacterial residue soaking solution in the pressure vessel was 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 organic solvent is ethyl acetate, and the pressure vessel pressure is ≥0.5 MPa.

3. 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 1) is ≥6h.

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

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

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

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

8. A method for extracting fusidic acid, comprising the preparation steps included in the method for extracting fusidic acid hemihydrate from fusidic acid bacterial residue as described in any one of claims 1-7.

Images