Preparation method of rapamycin

Abstract

The invention relates to the technical field of bioengineering, in particular to a rapamycin preparation method. The preparation method comprises the following steps: treating rapamycin through four fermentation stages based on fermentation temperature control to obtain rapamycin fermentation liquor, and separating and extracting the rapamycin fermentation liquor to obtain the rapamycin. According to the technical scheme provided by the embodiment of the invention, the rapamycin fermentation liquor is separated and extracted by controlling the fermentation process of the rapamycin through four stages, so that the rapamycin is obtained. The fermentation yield and purity of the rapamycin are obviously improved, and the production cost is greatly reduced.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and particularly relates to a method for preparing rapamycin. Background Art

[0002] Rapamycin is currently the most promising new and potent immunosuppressant in the world. Its molecular structure is similar to FK506 and it is another immunophilin binder. It can be used for anti-rejection in organ transplantation. Its immunosuppressive effect is dozens of times stronger than that of cyclosporine, and it is the immunosuppressant with the lowest nephrotoxicity. It can also be used to treat autoimmune diseases such as rheumatoid arthritis and lupus erythematosus. In September 1999, the US FDA officially approved rapamycin as an anti-rejection drug for kidney transplantation for the market. At the same time, the Cypher vascular stent coated with rapamycin developed by Cordis Corporation was successively launched in Europe, the United States and Japan in 2002. In terms of anti-tumor, the FDA approved its use in the treatment of renal cell carcinoma, mantle cell lymphoma, tuberous sclerosis and pancreatic cancer in 2007, 2008, 2010 and 2011 respectively, showing broad application prospects.

[0003] Hangzhou Zhongmei Huadong Pharmaceutical Co., Ltd. has put it into industrial production (Chinese Patent CN200810019207.8), and the fermentation ability of Streptomyces hygroscopicus cultured in 10-ton and 50-ton fermenters is about 600 mg / L. Zhang Wei et al. disclosed the strain selection and optimization of culture conditions in the article "Rational Breeding and New Fermentation Technology of High-yield Rapamycin-producing Strains" (Master's thesis of Zhejiang University in 2009), and the fermentation ability in a 20-ton fermenter was 700 mg / L.

[0004] In the patent "A Method for Increasing the Yield of Rapamycin by Streptomyces" (Chinese Patent CNCN202010084526) by Jiang Weihong, some target genes involved in the tricarboxylic acid cycle, fatty acid synthesis or aromatic amino acid synthesis pathways in the rapamycin-producing bacteria are closely related to the rapamycin yield of the producing bacteria. By constructing plasmids and transforming the constructed plasmids into Streptomyces rapamycinicus to down-regulate the expression of target genes in the rapamycin-producing bacteria, the recombinant expression system and the expression strain were optimized, and effective recombinant expression constructs were designed, so that the rapamycin expression levels reached 550 mg / L, 800 mg / L, and 750 mg / L. Using biotechnology to achieve yield increase is indeed a good method, which requires special equipment and specialized technical personnel, but it is not feasible for enterprises that do not have biotechnological conditions. At the same time, as is well known, the stability of plasmid transfection is not very good.

[0005] Zhao Zhiquan's "Method for Cultivating Streptomyces hygroscopicus to Produce Rapamycin Fermentation Broth" (Chinese Patent CN201010540111) obtains a fermentation broth with a relatively high concentration of rapamycin through the selection of the culture medium and the control of culture conditions, especially the control of the rotation speed. The titer of rapamycin in a 100L fermenter reaches about 900mg / L. Compared with genetic engineering technology and strain mutagenesis technology, controlling the fermentation process to increase the yield of rapamycin is more practical. Summary of the Invention

[0006] To solve the above problems, the embodiments of the present application provide a method for preparing rapamycin. By controlling very simple means such as the concentration of phosphate ions in the culture medium, the initial pH of the culture medium, and the control of fermentation parameters at specific time periods during the fermentation process, the fermentation yield and purity of rapamycin are significantly improved, and the production cost is greatly reduced.

[0007] To achieve the above object, the technical solutions adopted in the embodiments of the present application are as follows:

[0008] In the first aspect, the embodiments of the present application provide a method for preparing rapamycin. Based on the control of the fermentation temperature, the rapamycin strain is processed through four fermentation stages to obtain a rapamycin fermentation broth, and the rapamycin fermentation broth is separated and extracted to obtain rapamycin; the fermentation stages include the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage; the fermentation temperature in the first fermentation stage is 26-30°C, and the pH value of the fermentation broth at this time is the initial pH. The fermentation temperature in the second fermentation stage is 20-24°C, and the pH value of the fermentation broth decreases by 0.5-1 pH unit at this time. The fermentation temperature in the third fermentation stage is 25-27°C, and the pH value of the fermentation broth remains unchanged at this time. The fermentation temperature in the fourth fermentation stage is 27-29°C, and the pH value of the fermentation broth gradually rises to 7.2-7.5; the initial pH of the fermentation medium is 6.2-8.0.

[0009] Further, the fermentation environment in the four fermentation stages is a phosphate-containing fermentation medium.

[0010] Further, the concentration of phosphate ions in the fermentation medium is 0.005% - 0.05%.

[0011] Further, the dissolved oxygen content in the four fermentation stages is greater than or equal to 60%.

[0012] Further, the end mark of the first fermentation stage is: the concentration of the rapamycin bacteria in the fermentation broth is 10%, and there are bacterial particles with a size less than 0.1mm.

[0013] Further, the end mark of the second fermentation stage is: the concentration of the rapamycin bacteria in the fermentation broth is 20% - 25%, and the size of the bacterial particles develops to 0.5mm and above.

[0014] Further, the end mark of the third fermentation stage is that the concentration of Actinoplanes rapae in the fermentation broth is 30% - 35%, and the concentration of Actinoplanes rapae in the fermentation broth increases by 10% based on the second fermentation stage.

[0015] Further, the end mark of the fourth fermentation stage is that the cell concentration is maintained at the second fermentation stage.

[0016] In the technical solution provided by the embodiments of the present application, by controlling the four stages of the fermentation process of rapamycin, separating and extracting the rapamycin fermentation broth, rapamycin is obtained. The fermentation yield and purity of rapamycin are significantly improved, and the production cost is greatly reduced. Brief Description of the Drawings

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0018] Figure 1 It is a flow chart of the rapamycin preparation method provided by the embodiments of the present application. Detailed Embodiments

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase. The following described embodiments are some embodiments of the present invention, not all of the embodiments. Combining the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0020] In a specific embodiment of the present invention, the present invention provides a rapamycin preparation method, which divides the rapamycin fermentation process into four stages and controls the four stages. Among them, the rapamycin production strain in this embodiment is Actinoplanes sp. No902 - 109.

[0021] Among them, the fermentation environment for rapamycin is a fermentation medium containing phosphate, where the concentration of phosphate ions in the fermentation medium is 0.005% - 0.05%, the initial pH value of the fermentation medium is 6.2 - 8.0, and the dissolved oxygen content in the fermentation medium is greater than or equal to 60%.

[0022] Among them, the four stages in the embodiments of the present application are divided according to the change of the fermentation temperature. The fermentation temperature in the first fermentation stage is 26-30°C, the fermentation temperature in the second fermentation stage is 20-24°C, the fermentation temperature in the third fermentation stage is 25-27°C, and the fermentation temperature in the fourth fermentation stage is 27-29°C.

[0023] Among them, the above four fermentation stages respectively correspond to the four stages of the generation of the strain. Specifically, the first fermentation stage is the lag phase of the strain. In this fermentation stage, the strain recovers and grows slowly; the second fermentation stage is the rapid growth stage of the strain. In this stage, the strain enters the exponential growth phase; the third fermentation stage, in this stage, the strain still grows relatively fast, the cells begin to adhere to each other, the fermentation broth gradually becomes viscous, and the target substance begins to appear; the fourth fermentation stage, in this stage, the strain enters the stationary phase, turns to secondary metabolism mainly, and the target substance continuously increases. Through the fermentation of the above four stages, it enters the final decline phase. In this stage, the strain enters the decline phase and the target substance stops increasing. Among them, in the fourth fermentation stage, due to the loss of the bottom material of the culture medium, it is necessary to add a basic culture medium in the fourth fermentation stage.

[0024] And the above four fermentation stages are characterized by the following characteristic nodes in terms of data:

[0025] The end mark of the first fermentation stage is that the concentration of rapamycin bacteria in the fermentation broth is 10%, and at this time, the pH value of the fermentation broth does not change and remains the initial pH value.

[0026] The end mark of the second fermentation stage is that the concentration of rapamycin bacteria in the fermentation broth is 20%-25%, and at this time, the pH value of the fermentation broth drops to 5.2-7.0.

[0027] The end mark of the third fermentation stage is that the concentration of rapamycin bacteria in the fermentation broth is 30%-35%, and at this time, the pH value of the fermentation broth remains unchanged at the pH value in the second fermentation stage, which is 5.2-7.0.

[0028] The end mark of the fourth fermentation stage is that the concentration of rapamycin bacteria in the fermentation broth is the same as that in the third fermentation stage, and the pH value of the fermentation broth is greater than 7.5 and remains unchanged within a unit time. The unit time can be set specifically, and the unit time is the minimum value, that is, as long as the pH value remains unchanged within this time, the fermentation is completed.

[0029] Regarding the changes in the above fermentation stages, the state of the fermentation broth also changes. In the first fermentation stage, a large number of visible extremely fine bacterial grains appear in the fermentation broth, with the size of the bacterial grains being less than 0.1 mm. In the second fermentation stage, the fermentation broth becomes thick, and the bacterial grains in the fermentation broth can reach 0.5 mm or more. In the third fermentation stage, the fermentation broth significantly becomes viscous and forms a film on the wall. In the fourth fermentation stage, the viscosity of the fermentation broth decreases compared to the third fermentation stage.

[0030] Specifically, the specific fermentation steps in the embodiments of the present application are: plate seed culture - shake flask seed culture - seed tank seed culture - fermenter fermentation culture. Among them, the plate seed culture, shake flask seed culture, and seed tank seed culture all adopt conventional technical means in the art and will not be elaborated in the embodiments of the present application.

[0031] Among them, the culture environment in the embodiments of the present application is a basal medium containing phosphate. The basic fermentation medium is a conventional medium for microorganisms, especially actinomycetes, including a quick-acting carbon source such as glucose, a long-acting carbon source such as corn starch, one or several nitrogen sources such as cottonseed protein powder, yeast extract, corn steep liquor dry powder, etc. In addition, it also includes phosphate, calcium salt, and antifoaming agent. Among them, phosphate has an obvious influence on the growth of the bacteria. Usually, potassium dihydrogen phosphate and dipotassium hydrogen phosphate are used in combination, and the conventional dosage is 0.2% - 1% or even higher. Among them, the root ion that has an important influence on the production of rapamycin strains is the phosphate ion rather than the potassium ion. Within a certain range, as the concentration of the phosphate ion increases, the rapamycin yield linearly decreases. The suitable phosphate ion concentration for the bacteria in the embodiments of the present application is selected to be lower than the conventional phosphate dosage, and the specific phosphate ion concentration is 0.005% - 0.05%.

[0032] In the process of microbial growth and metabolism, it is divided into primary metabolism and secondary metabolism. Among them, primary metabolism mainly meets the needs of the microorganisms for their own reproduction, and secondary metabolism is often not necessary for the growth and reproduction of microorganisms and often changes from primary metabolism to secondary metabolism under unfavorable growth conditions. Fermentation precisely utilizes these two metabolic characteristics of microorganisms to obtain the fermentation products required by humans. To obtain a high yield, first, it is necessary to cultivate an appropriate number of microorganisms. Second, when an appropriate number of microorganisms are obtained, the external conditions need to be artificially controlled to break the metabolic control system of the microorganisms and make the microorganisms promptly transfer to secondary metabolism. Among them, the key to the high or low yield of rapamycin and the amount of impurities lies in controlling the growth rate in the second stage and the transformation from the third stage to the fourth stage. Among them, the yield can be further improved by adjusting the pH and temperature during the transformation process. And, to further ensure the stability of fermentation, a basal medium can optionally be supplemented into the system in the fourth fermentation stage.

[0033] Refer toFigure 1 , for the above fermentation process, the preparation method provided by the embodiments of the present application includes the following steps:

[0034] Step S1. Inoculate the rapamycin-producing bacterium into the basic fermentation medium.

[0035] In the embodiments of the present application, the rapamycin-producing bacterium is inoculated as Actinoplanes sp. No902-109. Among them, the concentration of phosphate ions in the basic fermentation medium is 0.005%, and the initial pH is 6.2.

[0036] Step S2. Fermentatively control the rapamycin-producing bacterium in the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage in sequence to obtain a fermentation broth.

[0037] In the embodiments of the present application, for the first fermentation stage, the culture temperature is 26-30 °C, for the second fermentation stage, the culture temperature is 20-24 °C, for the third fermentation stage, the culture temperature is 25-27 °C, and for the fourth fermentation stage, the culture temperature is 27-29 °C.

[0038] Step S3. Separate and extract the fermentation broth to obtain rapamycin.

[0039] The following further illustrates the rapamycin preparation method of the present invention through specific examples.

[0040] Example 1

[0041] S1. Inoculate Actinoplanes sp. No902-109 into the basic fermentation medium. The concentration of phosphate ions in the fermentation medium is 0.005%, and the initial pH is 6.2.

[0042] S2. Fermentatively control the rapamycin-producing bacterium in the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage in sequence to obtain a fermentation broth.

[0043] Among them, for the first fermentation stage, the culture temperature is 26 °C, for the second fermentation stage, the culture temperature is 20 °C, for the third fermentation stage, the culture temperature is 25 °C, and for the fourth fermentation stage, the culture temperature is 27 °C.

[0044] The total overall fermentation time is 240 h.

[0045] S3. After fermentation, a fermentation broth is obtained, and it is separated and extracted to obtain rapamycin.

[0046] In the embodiments of the present application, for the separation and extraction method, the rapamycin separation and extraction process in the prior art is adopted.

[0047] Example 2

[0048] S1. Inoculate Actinoplanes sp. No902 - 109 into the basic fermentation medium, where the phosphate ion concentration of the fermentation medium is 0.05% and the initial pH is 8.0.

[0049] S2. Ferment and control the rapamycin - producing bacteria in the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage in sequence to obtain the fermentation broth.

[0050] The culture temperature in the first fermentation stage is 30 °C, the culture temperature in the second fermentation stage is 22 °C, the culture temperature in the third fermentation stage is 27 °C, and the culture temperature in the fourth fermentation stage is 29 °C.

[0051] The total fermentation time is 240 h.

[0052] S3. After the fermentation is completed, obtain the fermentation broth and perform separation and extraction to obtain rapamycin.

[0053] In the embodiment of the present application, for the separation and extraction method, the rapamycin separation and extraction process in the prior art is adopted.

[0054] Example 3

[0055] S1. Inoculate Actinoplanes sp. No902 - 109 into the basic fermentation medium, where the phosphate ion concentration of the fermentation medium is 0.01% and the initial pH is 7.0.

[0056] S2. Ferment and control the rapamycin - producing bacteria in the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage in sequence to obtain the fermentation broth.

[0057] The culture temperature in the first fermentation stage is 28 °C, the culture temperature in the second fermentation stage is 24 °C, the culture temperature in the third fermentation stage is 26 °C, and the culture temperature in the fourth fermentation stage is 28 °C.

[0058] The total fermentation time is 240 h.

[0059] S3. After the fermentation is completed, obtain the fermentation broth and perform separation and extraction to obtain rapamycin.

[0060] In the embodiment of the present application, for the separation and extraction method, the rapamycin separation and extraction process in the prior art is adopted.

[0061] Comparative Example 1

[0062] S1. Inoculate Actinoplanes sp. No902 - 109 into the basic fermentation medium. The concentration of phosphate ions in the fermentation medium is 0.005%, and the initial pH is 6.2.

[0063] S2. Control the temperature at 26°C throughout the process, and the total fermentation time is 240 h.

[0064] S3. After fermentation, the obtained fermentation broth is separated and extracted to obtain rapamycin.

[0065] In this comparative example, for the separation and purification, the same prior art solution as in Examples 1 - 3 is adopted, and it will not be elaborated in the examples of this application.

[0066] Comparative Example 2

[0067] S1. Inoculate Actinoplanes sp. No902 - 109 into the basic fermentation medium. The concentration of phosphate ions in the fermentation medium is 0.005%, and the initial pH is 6.2.

[0068] S2. Control the fermentation temperature at 20°C throughout the process, and the total fermentation time is 240 h.

[0069] S3. After fermentation, the obtained fermentation broth is separated and extracted to obtain rapamycin.

[0070] In this comparative example, for the separation and purification, the same prior art solution as in Examples 1 - 3 is adopted, and it will not be elaborated in the examples of this application.

[0071] Comparative Example 3

[0072] S1. Inoculate Actinoplanes sp. No902 - 109 into the basic fermentation medium. The concentration of phosphate ions in the fermentation medium is 0.3%, and the initial pH is 6.2;

[0073] S2. Ferment and control the rapamycin - producing bacteria in the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage in sequence to obtain the fermentation broth.

[0074] Specifically, for the first fermentation stage, the culture temperature is 26°C, for the second fermentation stage, the culture temperature is 20°C, for the third fermentation stage, the culture temperature is 25°C, and for the fourth fermentation stage, the culture temperature is 27°C.

[0075] The total fermentation time is 240 h.

[0076] S3. After fermentation, the obtained fermentation broth is separated and extracted to obtain rapamycin.

[0077] In this comparative example, the same prior art solution as in Examples 1 to 3 is adopted for separation and purification, which will not be elaborated in the embodiments of the present application.

[0078] Comparative Example 4

[0079] S1. Inoculate Actinoplanes sp. No902-109 into the basic fermentation medium, where the phosphate ion concentration of the fermentation medium is 0.005% and the initial pH is 4.5;

[0080] S2. Ferment and control the rapamycin-producing bacteria in the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage in sequence to obtain the fermentation broth.

[0081] Specifically, for the first fermentation stage, the culture temperature is 26 °C, for the second fermentation stage, the culture temperature is 20 °C, for the third fermentation stage, the culture temperature is 25 °C, and for the fourth fermentation stage, the culture temperature is 27 °C.

[0082] The total fermentation time is 240 h.

[0083] S3. After fermentation, the obtained fermentation broth is separated and extracted to obtain rapamycin;

[0084] In this comparative example, the same prior art solution as in Examples 1 to 3 is adopted for separation and purification, which will not be elaborated in the embodiments of the present application.

[0085] Comparative Example 5

[0086] S1. Inoculate Actinoplanes sp. No902-109 into the basic fermentation medium, where the phosphate ion concentration of the fermentation medium is 0.05% and the initial pH is 8.0;

[0087] S2. Control the culture temperature at 30 °C throughout the process.

[0088] The total fermentation time is 240 h.

[0089] S3. After fermentation, the obtained fermentation broth is separated and extracted to obtain rapamycin.

[0090] In this comparative example, the same prior art solution as in Examples 1 to 3 is adopted for separation and purification, which will not be elaborated in the embodiments of the present application.

[0091] Comparative Example 6

[0092] S1. Inoculate Actinoplanes sp. No902-109 into the basic fermentation medium, where the phosphate ion concentration of the fermentation medium is 0.05% and the initial pH is 8.0;

[0093] S2. Control the culture temperature at 24 °C throughout the process.

[0094] The total fermentation time is 240 h.

[0095] S3. After the fermentation is completed, the obtained fermentation broth is separated and extracted to obtain rapamycin;

[0096] In this comparative example, the same prior art solution as in Examples 1 - 3 is adopted for separation and purification, which will not be elaborated in the embodiments of the present application.

[0097] Comparative Example 7

[0098] S1. Inoculate Actinoplanes sp. No902 - 109 into the basic fermentation medium. The phosphate ion concentration of the fermentation medium is 0.3%, and the initial pH is 8.0;

[0099] S2. Ferment and control the rapamycin - producing bacteria in the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage in sequence to obtain the fermentation broth.

[0100] Specifically, for the first fermentation stage, the culture temperature is 30 °C; for the second fermentation stage, the culture temperature is 22 °C; for the third fermentation stage, the culture temperature is 27 °C; for the fourth fermentation stage, the culture temperature is 29 °C.

[0101] The total fermentation time is 240 h.

[0102] S3. After the fermentation is completed, the obtained fermentation broth is separated and extracted to obtain rapamycin;

[0103] In this comparative example, the same prior art solution as in Examples 1 - 3 is adopted for separation and purification, which will not be elaborated in the embodiments of the present application.

[0104] Comparative Example 8

[0105] S1. Inoculate Actinoplanes sp. No902 - 109 into the basic fermentation medium. The phosphate ion concentration of the fermentation medium is 0.05%, and the initial pH is 4.5.

[0106] S2. Ferment and control the rapamycin - producing bacteria in the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage in sequence to obtain the fermentation broth.

[0107] Specifically, the culture temperature in the first stage is 30 °C; the culture temperature in the second stage is 22 °C; in the third stage, the culture temperature is 27 °C; in the fourth stage, the culture temperature is 29 °C.

[0108] The total fermentation time is 240 h.

[0109] S3. After fermentation is completed, the obtained fermentation broth is separated and extracted to obtain rapamycin;

[0110] In this comparative example, the same prior art solution as in Examples 1 - 3 is adopted for separation and purification, which will not be elaborated in the embodiments of the present application.

[0111] Comparative Example 9

[0112] S1. Inoculate Actinoplanes sp. No902 - 109 into the basic fermentation medium, where the phosphate ion concentration of the fermentation medium is 0.01% and the initial pH is 7.0.

[0113] S2. Control the culture temperature at 30°C throughout the process.

[0114] The total fermentation time is 240 h.

[0115] S3. After fermentation is completed, the obtained fermentation broth is separated and extracted to obtain rapamycin;

[0116] In this comparative example, the same prior art solution as in Examples 1 - 3 is adopted for separation and purification, which will not be elaborated in the embodiments of the present application.

[0117] Comparative Example 10

[0118] S1. Inoculate Actinoplanes sp. No902 - 109 into the basic fermentation medium, where the phosphate ion concentration of the fermentation medium is 0.01% and the initial pH is 7.0;

[0119] S2. Control the culture temperature at 22°C throughout the process.

[0120] The total fermentation time is 240 h.

[0121] S3. After fermentation is completed, the obtained fermentation broth is separated and extracted to obtain rapamycin;

[0122] In this comparative example, the same prior art solution as in Examples 1 - 3 is adopted for separation and purification, which will not be elaborated in the embodiments of the present application.

[0123] Comparative Example 11

[0124] S1. Inoculate Actinoplanes sp. No902 - 109 into the basic fermentation medium, where the phosphate ion concentration of the fermentation medium is 0.3% and the initial pH is 7.0.

[0125] S2. Ferment the rapamycin-producing bacteria in the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage in sequence to obtain a fermentation broth.

[0126] The culture temperature in the first stage is 28 °C; the culture temperature in the second stage is 24 °C; in the third stage, the culture temperature is 26 °C; in the fourth stage, the culture temperature is 28 °C.

[0127] The total fermentation time is 240 h.

[0128] S3. After the fermentation is completed, the obtained fermentation broth is separated and extracted to obtain rapamycin;

[0129] In this comparative example, the same prior art solution as in Examples 1 - 3 is adopted for separation and purification, and it will not be elaborated in the examples of this application.

[0130] Comparative Example 12

[0131] S1. Inoculate Actinoplanes sp. No902 - 109 in the basic fermentation medium, and the phosphate ion concentration of the fermentation medium is 0.01%, and the initial pH is 4.5.

[0132] S2. Ferment the rapamycin-producing bacteria in the first fermentation stage, the second fermentation stage, the third fermentation stage, and the fourth fermentation stage in sequence to obtain a fermentation broth.

[0133] Specifically, the culture temperature in the first fermentation stage is 28 °C, the culture temperature in the second fermentation stage is 24 °C, the culture temperature in the third fermentation stage is 26 °C, and the culture temperature in the fourth fermentation stage is 28 °C.

[0134] The total fermentation time is 240 h.

[0135] S3. After the fermentation is completed, the obtained fermentation broth is separated and extracted to obtain rapamycin;

[0136] In this comparative example, the same prior art solution as in Examples 1 - 3 is adopted for separation and purification, and it will not be elaborated in the examples of this application.

[0137] The differences between the above Examples 1 - 3 and Comparative Examples 1 - 12 are described as follows:

[0138] The differences between Comparative Examples 1 and 2 and Example 1 are that: during the whole fermentation and culture process, the temperature is not controlled in sections, but a fixed value is adopted for the temperature.

[0139] The difference between Comparative Example 3 and Example 1 is that: the phosphate ion concentration of the medium is inconsistent.

[0140] The difference between Comparative Example 4 and Example 1 lies in that the initial pH of the culture medium is inconsistent.

[0141] The differences between Comparative Example 5, Comparative Example 6 and Example 2 are that during the whole fermentation and culture process, the temperature is not controlled in sections, but a fixed value is adopted.

[0142] The difference between Comparative Example 7 and Example 2 lies in that the concentration of phosphate ions in the culture medium is inconsistent.

[0143] The difference between Comparative Example 8 and Example 2 lies in that the initial pH of the culture medium is inconsistent.

[0144] The differences between Comparative Example 9, Comparative Example 10 and Example 3 are that during the whole fermentation and culture process, the temperature is not controlled in sections, but a fixed value is adopted.

[0145] The difference between Comparative Example 11 and Example 3 lies in that the concentration of phosphate ions in the culture medium is inconsistent.

[0146] The difference between Comparative Example 12 and Example 3 lies in that the initial pH of the culture medium is inconsistent.

[0147] Experimental Example

[0148] For Examples 1 - 3 and Comparative Examples 1 - 12, blank group 1 and blank group 2 were set respectively to carry out fermentation to obtain fermentation results, and the fermentation results are shown in Table 1:

[0149] Table 1. Fermentation result table

[0150]

[0151] As can be seen from Table 1, for Comparative Example 3 and Example 1, Comparative Example 7 and Example 2, blank group 1 and Comparative Example 9, Comparative Example 11 and Example 3, the same culture temperature and pH value are adopted, and only the phosphate ions are different. When the fermentation reaches 240 h, the rapamycin yield of the latter is 1.34 times, 1.29 times, 1.21 times, 1.32 times that of the former; the proportion of rapamycin in the fermentation broth of the latter is 1.24 times, 1.22 times, 1.16 times, 1.20 times that of the former.

[0152] For Comparative Example 4 and Example 1, Comparative Example 8 and Example 2, Comparative Example 12 and Example 3, the same culture temperature and phosphate ions are adopted, and only the pH value is different. When the fermentation reaches 240 h, the rapamycin yield of the latter is 4.87 times, 4.78 times, 5.24 times that of the former; the proportion of rapamycin in the fermentation broth of the latter is 1.40 times, 1.45 times, 1.38 times that of the former.

[0153] For Comparative Examples 1 and 2 and Example 1, Comparative Examples 5 and 6 and Example 2, and Comparative Examples 9 and 10 and Example 3, the same pH value and phosphate ions were used, and only the culture temperature control was different. When the fermentation reached 240 h, the rapamycin yields of the latter were 1.22 times, 2.56 times, 1.20 times, 1.47 times, 1.23 times, and 2.39 times that of the former; the proportions of rapamycin in the fermentation broth of the latter were 1.14 times, 1.31 times, 1.11 times, 1.27 times, 1.15 times, and 1.35 times that of the former.

[0154] Regarding Example 1, Example 2, and Example 3, the culture temperature was controlled in segments, and the pH value and phosphate ions were also within the controlled range. Compared with Comparative Examples 1 - 12 which only controlled any two of them, when the fermentation reached 240 h, the combined control of the three factors resulted in significantly higher rapamycin yields and proportions of rapamycin in the fermentation broth than the control of any two of them. This shows that the fermentation parameters are not set randomly, which is related to the metabolic mechanisms at different growth stages of the strain. The three factors do not achieve good results by simple setting, but require the coordinated and reasonable combination of the factor parameters. The solution provided by the present invention enables the three factors to achieve a true synergistic effect, affecting microbial fermentation, and ultimately achieving the purpose of significantly increasing the rapamycin yield and the proportion of rapamycin in the fermentation broth.

[0155] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing rapamycin, characterized in that: Based on the fermentation temperature control, the rapamycin strain is processed through four fermentation stages to obtain the rapamycin fermentation liquid, and the rapamycin fermentation liquid is separated and extracted to obtain rapamycin; the fermentation stages include the first fermentation stage, the second fermentation stage, the third fermentation stage and the fourth fermentation stage; the fermentation temperature of the first fermentation stage is 26-30°C, at which time the pH value of the fermentation liquid is the initial pH, the fermentation temperature of the second fermentation stage is 20-24°C, at which time the pH value of the fermentation liquid decreases by 0.5-1 pH units, the fermentation temperature of the third fermentation stage is 25-27°C, at which time the pH value of the fermentation liquid remains unchanged, the fermentation temperature of the fourth fermentation stage is 27-29°C, and the pH value of the fermentation liquid gradually increases to 7.2-7.5; the initial pH of the fermentation medium is 6.2-8.

0.

2. The method for preparing rapamycin according to claim 1, characterized in that: The fermentation environment of the four fermentation stages is a phosphate-containing fermentation medium.

3. The method for preparing rapamycin according to claim 2, characterized in that: The phosphate ion concentration of the fermentation medium is between 0.005% and 0.05%.

4. The method for preparing rapamycin according to claim 1, characterized in that: The dissolved oxygen content in the four fermentation stages is greater than or equal to 60%.

5. The method for preparing rapamycin according to claim 1, characterized in that: The end mark of the first fermentation stage is: the concentration of rapamycin in the fermentation liquid is 10%, and bacterial particles with a size less than 0.1 mm appear.

6. The method for preparing rapamycin according to claim 1, characterized in that: The end mark of the second fermentation stage is: the concentration of rapamycin in the fermentation liquid is 20% to 25%, and the size of the bacterial particles develops to 0.5 mm or above.

7. The method for preparing rapamycin according to claim 1, characterized in that: The end mark of the third fermentation stage is: the rapamycin concentration in the fermentation broth is 30% to 35%, and the rapamycin concentration in the fermentation broth is increased by 10% based on the second fermentation stage.

8. The method for preparing rapamycin according to claim 1, characterized in that: The end mark of the fourth fermentation stage is: the bacterial concentration is maintained at the second fermentation stage.

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