Production method of high-yield prodigiosin extract

Abstract

The invention relates to the technical field of fermentation engineering, and provides a high-yield prodigiosin extract production method, which comprises: S1, preparing a mother strain; s2, preparing a seed solution; s3, primary fermentation culture; s4, carrying out productive multi-stage fermentation culture; s5, extracting a prodigiosin crude extract; and S6, purifying the prodigiosin. In the production process, specific serratia marcescens is selected for activation, optimization and re-fermentation, a multi-stage productive fermentation mode is adopted, and the treated prodigiosin crude extract is purified by using a column chromatography separation technology and a high-pressure liquid phase instrument, so that the content of the obtained prodigiosin is high.

Description

A method for producing high-yield erythromycin extract Technical Field

[0001] This invention belongs to the field of fermentation engineering technology, specifically relating to a method for producing high-yield erythromycin extract. Background Technology

[0002] As a natural pigment of significant value, styrax has shown broad application prospects in multiple fields such as food, medicine, and cosmetics due to its excellent coloring properties, antioxidant activity, and potential bioactivity, and market demand continues to grow.

[0003] In the food industry, with the increasing demand for natural and healthy foods, styraxanthin, with its safe, non-toxic, strong coloring power, and high stability, can be widely used for coloring various foods, such as beverages, pastries, and sauces. This effectively enhances the appearance and market competitiveness of food products, meeting consumers' expectations for natural food additives. In the pharmaceutical field, the antioxidant, antibacterial, and antiviral bioactivities of styraxanthin have garnered significant attention in drug development and health food development, providing new ideas and methods for the prevention and treatment of related diseases. In the cosmetics industry, the antioxidant effects of styraxanthin help delay skin aging and improve skin condition. It can be used as a natural antioxidant and colorant in skincare and makeup products, satisfying people's pursuit of natural and green cosmetics.

[0004] However, the production of styraxin still faces many challenges. Traditional extraction methods mostly obtain it from the fermentation products of the microorganisms that produce the pigment, but these methods suffer from low yields, high costs, and complex extraction processes, making it difficult to meet the needs of large-scale industrial production. While some chemical synthesis methods can increase yields to some extent, they are often accompanied by drawbacks such as environmental pollution, demanding synthesis conditions, and low product purity, limiting their further application and development.

[0005] Chinese patent CN 118667699 A discloses a fermentation medium for high-yield styraxin and a method for producing styraxin. The fermentation medium consists of the following components and proportions: 2-4 g / L peanut powder, 10-20 g / L peptone, and 1-5 g / L (MgSO4 + CaCl2) at a mass ratio of 49:1; peanut oil (1%-6%) and glycerol (1%-3%) are also added according to the volume percentage of the fermentation medium. While this method improves the yield and purity of styraxin by optimizing the medium composition, it does not fully consider the fine-tuning of fermentation and purification process parameters, leaving room for improvement in fermentation efficiency and yield.

[0006] Therefore, there is an urgent need to develop a high-yield production method for styraxone to improve its quantity and quality and promote its widespread application in various industries. Summary of the Invention

[0007] To address the existing technical problems, the present invention aims to provide a high-yield method for producing squalene. In this invention, specific *Serratia marcescens* strains are activated, optimized, and then fermented. A multi-stage productive fermentation process is employed, followed by column chromatography to purify the crude squalene extract, resulting in a high squalene content.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The present invention provides a method for producing high-yield strychnine extract, comprising the following steps: S1. Preparation of mother culture: After activating Serratia marcescens, under aseptic conditions, the strain is inoculated onto a medium containing a mixture of 1-3 wt% LB broth and 1-3 wt% agar powder, and cultured in a constant temperature incubator at 27-29℃ for 22-26 hours. After the bright red strain grows, the dark red strain is selected and streaked onto a medium containing 1-3 wt% LB broth. S1. Incubate the yeast culture medium obtained in step S1 in a petri dish containing a mixture of broth and 1-3 wt% agar powder for 22-26 hours, and select the strain with the brightest red color as the starter yeast strain; S2. Prepare the seed culture: Under aseptic conditions, inoculate the starter yeast strain obtained in step S1 into a culture medium containing 1-3 wt% LB broth, and incubate in a 27-29℃ full-temperature shaking incubator at a speed of 160-200 r / min for 16-20 hours to obtain the seed culture; S3. Preliminary fermentation culture: Measure the amount of the seed culture obtained in step S2... S3. Add the fermented liquid to the liquid culture medium and culture in a 27-29℃ full-temperature shaking incubator at a speed of 160-200 r / min for 22-26 h to obtain a fermentation broth containing styraxin; S4. Production-scale multi-stage fermentation culture: Use the fermentation broth containing styraxin obtained in step S3 as the mother liquor and carry out multi-stage fermentation in a liquid culture medium to obtain a fermentation broth; S5. Extraction of crude styraxin extract: The fermentation broth obtained in step S4 is crushed, centrifuged, and the centrifuged liquid is subjected to primary single-effect concentration. Reduce the liquid to remove 90-95% of the water, and then concentrate the remaining liquid to a paste. Mix the concentrated fermentation broth with a compound organic solvent, stir to dissolve, and let stand for 22-26 hours. Extract the supernatant, perform secondary separation, filtration, fine filtration, and rotary evaporation to obtain crude extract of squalene. S6. Purify squalene: Dissolve the crude extract of squalene obtained in step S5 in methanol, separate and purify it using silica gel column chromatography, and then separate and purify it using high-performance liquid chromatography to obtain squalene extract.

[0009] The reaction mechanism and function of the present invention are as follows: 1. After selecting Serratia marcescens, the present invention performs secondary optimization of the strain during the activation process. In a specific culture medium, the strain is continuously optimized to make the yeast strain more active and more capable of utilizing and transforming the substrate. Using it as a mother culture can provide a high-yield strain basis for the subsequent fermentation process, ensuring a high yield of styraxin from the source.

[0010] 2. The liquid culture medium and liquid culture medium in steps S3-S4 of this invention have unique and carefully optimized compositions, containing a variety of nutrients and special additives (such as Tween and antifoaming agents). Tryptone, glycerol, and other carbon and nitrogen sources provide basic energy and raw materials for cell growth and synthesis. The addition of Tween improves the dispersibility of nutrients in the culture medium and the accessibility of nutrients to the cells, especially for hydrophobic components like soybean oil, where Tween allows for better utilization by the cells, promoting metabolic pathways related to the synthesis of styraxone. Furthermore, the addition of antifoaming agents in productive multi-stage fermentation culture solves common foaming problems during fermentation, ensuring stable operation of the fermentation process and improving fermentation efficiency.

[0011] The synergistic optimization of multiple components in this invention fully considers the comprehensive nutritional requirements for cell growth and styraxin synthesis, as well as the physicochemical environment of the fermentation process. This is an innovative manifestation of the culture medium formulation in this process, which helps to improve the yield and quality of styraxin.

[0012] 3. This invention employs three fermenters for multi-stage fermentation cultivation, with precise control over fermentation parameters such as pH, temperature, rotation speed, pressure, and dissolved oxygen (DO) at each stage, and gradually scaling up the fermentation scale. This multi-stage fermentation cultivation fully considers the phased requirements of cell growth and product synthesis, the adaptability of the cells to the fermentation environment, and the stability and repeatability of the fermentation process, thereby achieving efficient production of styraxanthin.

[0013] 4. In the extraction of crude extract of styraxone, this invention employs a series of complex and precise operational steps, including homogenization and crushing, disc centrifugation, two-stage concentration, mixing and dissolving in a compound organic solvent and allowing to stand, ceramic membrane filtration, and ultrafine nano-scale ceramic membrane filtration. During purification, silica gel column chromatography is used for separation and purification, and specific elution and evaporation conditions are employed. Only by combining all these special treatments can a styraxone product with high purity and few impurities be finally obtained.

[0014] Specifically, in step S5, a composite organic solvent (ethyl acetate and acidified methanol) is used to extract styraxin, and the volume ratio of the concentrated fermentation broth, ethyl acetate, and acidified methanol is adjusted. By selecting the combination of ethyl acetate and acidified methanol, and utilizing their different polarities and interactions with styraxin molecules, better dissolution and separation effects are achieved, providing higher-quality raw materials for subsequent purification steps, thereby improving the feasibility and economy of the entire production process.

[0015] In some embodiments, the liquid culture medium in step S3, per 1L, comprises the following components: 1-3 g / L tryptone, 0.1-2 g / L glycerol, 0.5-3 g / L Tween, 0.1-2 g / L sodium chloride, 0.1-2 g / L potassium chloride, 0.1-2 g / L sodium acetate, 1-5 g / L soybean oil, with the remainder being purified water.

[0016] In some embodiments, the amount of seed liquid added in step S3 is 8-12% of the volume percentage of the liquid culture medium.

[0017] In some embodiments, the liquid culture medium in step S4, per 1L, comprises the following components: 1-3 g / L tryptone, 0.1-1.0 g / L glycerol, 0.1-2.0 g / L Tween, 0.1-2 g / L sodium chloride, 0.1-2 g / L potassium chloride, 0.1-2 g / L sodium acetate, 0.1-2 g / L defoamer, 1-5 g / L soybean oil, with the balance being purified water.

[0018] In some embodiments, the specific steps of the multi-stage fermentation in step S4 are as follows: S41. The fermentation broth containing styracil obtained in step S3 is added as the mother liquor to fermenter 1, and aerated fermentation is carried out in a liquid culture medium at a pH of 7.0-7.5, a temperature of 27-29℃, a rotation speed of 180-220 r / min, a pressure of 0.11-0.13 MPa, and a dissolved oxygen (DO) control of 2-2.2 m³ / min. 3 Fermentation is carried out at a constant temperature for 38-42 hours to obtain fermentation broth 1; S42. Fermentation broth 1 obtained in step S41 is transferred to fermenter 2 and further fermented in liquid culture medium with aeration, pH 7.0-7.5, temperature 27-29℃, rotation speed 160-200 r / min, pressure 0.11-0.13 MPa, and DO controlled at 18-22 mg / L. 3 Fermentation is carried out at a constant temperature for 38-42 hours to obtain fermentation broth 2; S43. Fermentation broth 2 obtained in step S42 is transferred to fermenter 3 and further fermented in liquid culture medium with aeration, pH value of 7.0-7.5, temperature of 27-29℃, rotation speed of 160-200 r / min, pressure of 0.11-0.13 MPa, and DO controlled at 145-155 mg / L. 3 Fermentation time is 38-42 hours per hour to obtain fermentation broth.

[0019] In some embodiments, the amount of fermentation broth containing styraxin added in step S4 is 8-12% of the volume percentage of the liquid culture medium.

[0020] In some embodiments, the composite organic solvent in step S5 is composed of ethyl acetate and acidified methanol.

[0021] Preferably, the acidified methanol is composed of hydrochloric acid and methanol in a volume ratio of 1:5.

[0022] In some embodiments, the volume ratio of the concentrated fermentation broth, ethyl acetate, and acidified methanol in step S5 is 1:(0.8-1.2):(0.9-1.1).

[0023] Preferably, the volume ratio of the concentrated fermentation broth, ethyl acetate, and acidified methanol in step S5 is 1:1:1.

[0024] In some embodiments, the silica gel column chromatography separation and purification in step S6 uses Sephadex LH-20 gel packing.

[0025] In some embodiments, the extract obtained by the production method contains >98% styracin.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. In the production process, the present invention selects specific Serratia marcescens for activation, optimization and re-fermentation, and adopts a multi-stage production fermentation method. Then, column chromatography separation technology is used to purify the crude extract of serotonin, so that the obtained serotonin content is high.

[0027] 2. The liquid culture medium and liquid culture medium in steps S3 to S4 of the present invention have unique and carefully optimized compositions, containing a variety of nutrients and special additives (such as Tween, defoamers, etc.). The synergistic optimization of multiple components ensures the stable operation of the fermentation process and improves fermentation efficiency.

[0028] 3. This invention employs three fermenters to sequentially conduct multi-stage fermentation culture. The multi-stage fermentation culture fully considers the staged requirements of cell growth and product synthesis, the adaptability of the cells to the fermentation environment, and the stability and repeatability of the fermentation process, thereby achieving efficient production of styraxin.

[0029] 4. In the extraction of crude extract of styraxone, this invention uses a compound organic solvent (ethyl acetate and acidified methanol) to extract styraxone, and adjusts the volume ratio of the concentrated fermentation broth, ethyl acetate and acidified methanol to achieve better dissolution and separation, providing higher quality raw materials for subsequent purification steps. Detailed Implementation

[0030] The present invention will be described below with reference to specific embodiments. It should be noted that the following embodiments are examples of the present invention and are used only to illustrate the invention, not to limit it. Other combinations and various modifications within the scope of the present invention can be made without departing from its spirit or scope.

[0031] According to the following examples and comparative examples, the proportions and processes of each raw material are specified to produce styrax red.

[0032] To facilitate the implementation of this invention by those skilled in the art, the manufacturers of some raw materials in the embodiments and comparative examples are described as follows: the defoamer is model YL-334; Serratia marcescens is deposited at the China General Microbiological Culture Collection Center, accession number CGMCC No. 5230; other raw materials are not specifically mentioned and can be purchased from the market.

[0033] Example 1 S1. Preparation of Mother Culture: After activating *Serratia marcescens*, the strain was inoculated into a mixture of 2 wt% LB broth and 1.8 wt% agar powder under aseptic conditions and cultured in a constant temperature incubator at 28℃ for 24 hours. After the growth of bright red strains, the dark red strains were selected and streaked onto a Petri dish containing the mixture of 2 wt% LB broth and 1.8 wt% agar powder, and cultured for another 24 hours. The strain with the brightest red color was selected as the starter culture. S2. Preparation of Seed Culture: Under aseptic conditions, the starter culture obtained in step S1 was inoculated into a culture medium containing 2 wt% LB broth and cultured in a constant temperature shaking incubator at 28℃ and a rotation speed of 180 r / min for 18 hours to obtain the seed culture. S3. Preliminary Fermentation Culture: 20 mL of the seed culture obtained in step S2 was added to 200 mL of liquid culture medium, with a pH of 7.0, and fermented at 28℃. In a full-temperature shaking incubator, the rotation speed was 180 r / min for 24 h to obtain a fermentation broth containing styraxin. The liquid culture medium contained the following components per 1 L: tryptone 1.5 g / L, glycerol 0.5 g / L, Tween 1.0 g / L, sodium chloride 0.3 g / L, potassium chloride 0.2 g / L, sodium acetate 0.3 g / L, soybean oil 3 g / L, with the remainder being purified water. S4. Production-scale multi-stage fermentation culture: The fermentation broth containing styraxin obtained in step S3 was used as the mother liquor for multi-stage fermentation in a liquid culture medium to obtain the fermentation broth. The specific steps of the multi-stage fermentation are as follows: S41. The fermentation broth containing styraxin obtained in step S3 was added to fermenter 1 as the mother liquor and fermented under aeration in a liquid culture medium at a pH of 7.0, a temperature of 28℃, a rotation speed of 200 r / min, a pressure of 0.12 MPa, and DO controlled at 2 mg / L. 3 / h, ferment for 40h to obtain fermentation broth 1; S42. Transfer fermentation broth 1 obtained in step S41 to fermenter 2, and further ferment in liquid culture medium with aeration, pH 7.0, temperature 28℃, rotation speed 180r / min, pressure 0.12MPa, DO controlled at 20m 3 / h, ferment for 40h to obtain fermentation broth 2; S43. Transfer fermentation broth 2 obtained in step S42 to fermenter 3, and further ferment in liquid culture medium with aeration, pH value 7.0, temperature 28℃, rotation speed 180r / min, pressure 0.12MPa, DO controlled at 150m 3 / h, fermentation for 40h to obtain fermentation broth; the liquid culture medium contains the following components: tryptone 1.5g / L, glycerol 0.5g / L, Tween 1.0g / L, sodium chloride 0.3g / L, potassium chloride 0.2g / L, sodium acetate 0.3g / L, defoamer 0.5g / L, soybean oil 3g / L; S5. Extraction of crude extract of styraxone: The fermentation broth obtained in step S4 is crushed in a homogenizer at a working pressure of 40MPa, and the crushed liquid is then transferred to a disc centrifuge for preliminary separation. The centrifuged liquid is subjected to one... First-stage single-effect concentration: The centrifuged liquid is concentrated at 80℃ until 95% of the water is removed. The remaining liquid is then transferred to a spherical scraper concentrator for secondary concentration at 60℃ until a paste is formed. The concentrated fermentation broth is then mixed with ethyl acetate and acidic methanol at a volume ratio of 1:1:1. After stirring to dissolve, the mixture is allowed to stand for 24 hours. The supernatant is then collected and subjected to secondary separation. The mixture is filtered through a ceramic membrane filter to remove insoluble impurities, followed by fine filtration through an ultrafine nano-sized ceramic membrane filter. The remaining liquid is then subjected to rotary evaporation at 42℃ and a rotation speed of 400 rpm. S5. Purification of 15g of the crude styracin extract obtained in step S5: Dissolve 15g of the crude styracin extract obtained in step S5 in 40mL of methanol, then transfer it to a 50mL centrifuge tube. Centrifuge at 7000rpm for 10min. Using a Sephadex LH-20 gel column, when the mobile phase liquid level is approximately 0.5cm above the gel interface, gently add the centrifuged sample dropwise along the inner wall of the column using a 5mL pipette. After the addition is complete, open the stopcock at full speed. When the added sample flows along the inner wall of the column to within 0.5cm of the gel interface... When the stopcock is closed, add sufficient methanol to the chromatography column, open the stopcock at the bottom of the column, collect the red eluent fraction and put it into a round-bottom flask. Install the flask on a rotary evaporator and evaporate it at a temperature of 42℃, a rotation speed of 40 rpm, and a pressure of 0.08 MPa. Then dissolve the paste with as little methanol as possible, and then use a high-performance liquid chromatography instrument for separation and purification to obtain the strychnine extract.

[0034] Example 2 A method for producing high-yield erythromycin extract, the specific implementation method is the same as in Example 1, except that the amount of seed liquid added in step S3 is 7% of the volume percentage of the liquid culture medium.

[0035] Example 3A method for producing high-yield erythromycin extract, the specific implementation method is the same as in Example 1, the difference being that the liquid culture medium in step S4 contains the following components: tryptone 1.5 g / L, glycerol 0.5 g / L, Tween 1.0 g / L, sodium chloride 0.3 g / L, potassium chloride 0.2 g / L, sodium acetate 0.3 g / L, and soybean oil 3 g / L.

[0036] Example 4 A method for producing high-yield lecithin extract, the specific implementation method is the same as in Example 1, the difference being that the amount of fermentation broth containing lecithin added in step S4 is 7% of the volume percentage of the liquid culture medium.

[0037] Example 5 A method for producing high-yield erythromycin extract, the specific implementation method is the same as in Example 1, the difference being that in step S5, the concentrated fermentation broth is mixed with ethyl acetate and acidic methanol at a volume ratio of 1:0.7:1 (the acidic methanol is composed of hydrochloric acid and methanol at a volume ratio of 1:5).

[0038] Example 6 A method for producing high-yield erythromycin extract, the specific implementation method is the same as in Example 1, the difference being that in step S5, the concentrated fermentation broth is mixed with ethyl acetate and acidic methanol at a volume ratio of 1:1:0.7 (the acidic methanol is composed of hydrochloric acid and methanol at a volume ratio of 1:5).

[0039] Example 7 A method for producing high-yield erythromycin extract, the specific implementation method is the same as in Example 1, the difference being that in step S6, the silica gel column chromatography separation and purification is carried out using Sephadex G-10 gel packing.

[0040] Comparative Example 1 A method for producing high-yield styraxin extract, the specific implementation method is the same as in Example 1, the difference being step S4: the fermentation broth containing styraxin obtained in step S3 is added as the mother liquor to the fermenter, and aerated fermentation is carried out in a liquid culture medium at a pH of 7.0, a temperature of 28℃, a rotation speed of 200 r / min, a pressure of 0.12 MPa, and a dissolved oxygen (DO) control of 20 mg / L. 3 Fermentation time was 120 hours, and the fermentation liquid was obtained.

[0041] Comparative Example 2 A method for producing high-yield erythromycin extract is the same as in Example 1, except that the organic solvent in step S5 is acidic methanol (composed of hydrochloric acid and methanol in a volume ratio of 1:5).

[0042] The content of styraxin was determined by HPLC using the extracts obtained from Examples 1-7 and Comparative Examples 1-2. The specific results are shown in Table 1.

[0043] Table 1

[0044] The results in Table 1 show that the squalene content obtained in Example 1 is high, indicating good application value. Compared to Example 1, Example 2 changed the amount of seed culture added to the liquid culture medium in step S3; Example 3 changed the composition of the liquid culture medium in step S4, omitting the addition of an antifoaming agent; Example 4 changed the proportion of squalene fermentation broth added to the liquid culture medium in step S4 compared to Example 1. Although these changes in the fermentation process maintained the squalene content above 98%, it was still somewhat lower than in Example 1. Combining Comparative Example 1 and Example 1, multi-stage fermentation culture helps to increase the fermentation degree, resulting in a significant increase in content.

[0045] Compared to Example 1, Examples 5-6 changed the volume ratio of the concentrated fermentation broth to ethyl acetate and acidic methanol; Example 7 changed the gel used in the silica gel column chromatography separation and purification in step S6, replacing Sephadex LH-20 gel packing with Sephadex G-10 gel packing; Comparative Example 2 changed the type of organic solvent in step S5 compared to Example 1; As can be seen from the data in the table, the adjustment of the purification steps will ultimately affect the content of styraxin.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present application in any way. Although the present application discloses the preferred embodiment as described above, it is not intended to limit the present application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of the present application using the disclosed technical content are equivalent to equivalent implementation cases. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the technical solution of the present invention are still within the scope of the technical solution.

Claims

1. A method for producing high-yield erythromycin extract, characterized in that, The process includes the following steps: S1. Preparation of mother culture: After activating Serratia marcescens, the strain is inoculated into a medium containing a mixture of 1-3 wt% LB broth and 1-3 wt% agar powder under aseptic conditions. The culture is then incubated at 27-29℃ for 22-26 hours. Once a bright red strain has grown, the dark red strain is selected and streaked onto a petri dish containing the mixture of 1-3 wt% LB broth and 1-3 wt% agar powder. The culture is continued for another 22-26 hours, and the brightest red strain is selected as the starter culture. S2. Preparation of seed culture: Under aseptic conditions, the starter culture obtained in step S1 is inoculated into a medium containing 1-3 wt% LB broth and incubated at 27-29℃ in a constant temperature shaking incubator at 160-200 r / min for 16-20 hours to obtain the seed culture. S3. Preliminary fermentation culture: Measure the seed liquid obtained in step S2 and add it to the liquid culture medium. In a full-temperature shaking incubator at 27-29℃, the rotation speed is 160-200 r / min, and the culture is carried out for 22-26 h to obtain a fermentation broth containing styraxin. S4. Production-scale multi-stage fermentation culture: The fermentation broth containing squalene obtained in step S3 is used as the mother liquor and fermented in a liquid culture medium in multiple stages to obtain the fermentation broth; S5. Extraction of crude squalene extract: The fermentation broth obtained in step S4 is crushed, centrifuged, and the centrifuged liquid is concentrated in a single-effect manner to remove 90-95% of the water. The remaining liquid is concentrated again to a paste. The concentrated fermentation broth is mixed with a compound organic solvent, stirred and dissolved, and allowed to stand for 22-26 hours. The supernatant is extracted, and the mixture is separated, filtered, finely filtered, and rotary evaporated to obtain the crude squalene extract; S6. Purification of squalene: The crude squalene extract obtained in step S5 is dissolved in methanol, separated and purified by silica gel column chromatography, and then separated and purified by high-performance liquid chromatography to obtain the squalene extract.

2. The method for producing a high-yield extract of erythromycin according to claim 1, characterized in that, In step S3, the liquid culture medium, per 1L, contains the following components: tryptone 1-3g / L, glycerol 0.1-2g / L, Tween 0.5-3g / L, sodium chloride 0.1-2g / L, potassium chloride 0.1-2g / L, sodium acetate 0.1-2g / L, soybean oil 1-5g / L, and the remainder is purified water.

3. The method for producing a high-yield extract of erythromycin according to claim 2, characterized in that, The amount of seed liquid added in step S3 is 8-12% of the volume percentage of the liquid culture medium.

4. The method for producing a high-yield extract of erythromycin according to claim 1, characterized in that, In step S4, the liquid culture medium, per 1L, contains the following components: tryptone 1-3g / L, glycerol 0.1-1.0g / L, Tween 0.1-2.0g / L, sodium chloride 0.3g / L, potassium chloride 0.1-2g / L, sodium acetate 0.1-2g / L, defoamer 0.1-2g / L, soybean oil 1-5g / L, with the remainder being purified water.

5. The method for producing a high-yield extract of erythromycin according to claim 1, characterized in that, The specific steps of the multi-stage fermentation described in step S4 are as follows: S41. The fermentation broth containing styracil obtained in step S3 is added as the mother liquor to fermenter 1, and aerated fermentation is carried out in liquid culture medium at a pH of 7.0-7.5, a temperature of 27-29℃, a rotation speed of 180-220 r / min, a pressure of 0.11-0.13 MPa, and a dissolved oxygen (DO) level of 2-2.2 mM. 3 Fermentation is carried out at a constant temperature for 38-42 hours to obtain fermentation broth 1; S42. Fermentation broth 1 obtained in step S41 is transferred to fermenter 2 and further fermented in liquid culture medium with aeration, pH 7.0-7.5, temperature 27-29℃, rotation speed 160-200 r / min, pressure 0.11-0.13 MPa, and DO controlled at 18-22 mg / L. 3 Fermentation is carried out at a constant temperature for 38-42 hours to obtain fermentation broth 2; S43. Fermentation broth 2 obtained in step S42 is transferred to fermenter 3 and further fermented in liquid culture medium with aeration, pH value of 7.0-7.5, temperature of 27-29℃, rotation speed of 160-200 r / min, pressure of 0.11-0.13 MPa, and DO controlled at 145-155 mg / L. 3 Fermentation time is 38-42 hours per hour to obtain fermentation broth.

6. The method for producing a high-yield extract of erythromycin according to claim 1, characterized in that, The amount of fermentation broth containing styraxin added in step S4 is 8-12% of the volume percentage of the liquid culture medium.

7. The method for producing a high-yield extract of erythromycin according to claim 1, characterized in that, The composite organic solvent in step S5 is composed of ethyl acetate and acidified methanol.

8. The method for producing a high-yield styrax erythrin extract according to claim 7, characterized in that, The volume ratio of the concentrated fermentation broth, ethyl acetate, and acidified methanol in step S5 is 1:(0.8-1.2):(0.9-1.1).

9. The method for producing a high-yield extract of erythromycin according to claim 1, characterized in that, In step S6, the silica gel column chromatography separation and purification process uses Sephadex LH-20 gel packing.

10. A method for producing a high-yield styrax erythrin extract according to any one of claims 1-9, characterized in that, The extract obtained by the production method contains >98% styracin.

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