A fermentation medium capable of increasing the yield of avermectin

Abstract

The present invention relates to a fermentation medium capable of increasing the yield of avermectin, and belongs to the technical field of microbial fermentation medium. The fermentation medium of the present invention comprises a basic culture solution and a replenishing solution, wherein the basic culture solution comprises 12-15 g / L of peanut meal extract I, 20-25 g / L of peanut meal biochar, 20-30 g / L of gelatinized potato starch, 8-10 g / L of yeast extract powder, 0.3-0.5 g / L of magnesium sulfate heptahydrate, 0.3-0.5 g / L of diammonium hydrogen phosphate, and 1-1.2 g / L of calcium lactate; the replenishing solution comprises 15-18 g / L of peanut meal extract II, 10-12 g / L of erythritol, 3-5 g / L of potassium chloride, 10-12 g / L of ammonium sulfate, and 5-8 g / L of brown algae polysaccharide. When the culture medium provided in the present invention is used for avermectin fermentation production, the yield of avermectin can be significantly increased, and the product has certain stability.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation medium and relates to a fermentation medium capable of improving the yield of avermectin. Background Art

[0002] Avermectin is a widely used agricultural or veterinary fungicide, insecticide, and acaricide, also known as amethin. Avermectin is a type of 16-membered macrolide compound with fungicidal, insecticidal, acaricidal, and nematicidal activity first developed by Satoshi Omura of Kitasato University in Japan and Merck Company in the United States. It is produced by fermentation of Streptomyces avermitilis in Streptomyces.

[0003] Fermentation medium is an important platform for the industrialization of fermentation products, which will directly affect the production and cost of avermectin. Nowadays, there are many types of culture media used for industrial fermentation. The components in the culture medium, especially the natural components, have a great influence on the fermentation yield. Due to the growth characteristics of Streptomyces avermitilis, only batch fermentation can be used as the main production method, resulting in large differences in the production of avermectin in different fermenters due to strain degradation, different lag phases, and changes in carbon and nitrogen source flow requirements. This difference limits the extraction and purification of avermectin to a certain extent and increases production costs. Therefore, it is very important to choose a better culture medium. Summary of the invention

[0004] In order to increase the yield of avermectin, the present invention provides a culture medium which can effectively improve fermentation efficiency, reduce cost consumption, and increase the yield of avermectin. The culture medium can realize stable and efficient production of avermectin.

[0005] The present invention adopts the following technical solutions to achieve the above purpose:

[0006] A fermentation medium comprises a basic culture solution and a supplementary solution, wherein the basic culture solution comprises 12-15 g / L of peanut meal extract I, 20-25 g / L of peanut meal biochar, 20-30 g / L of gelatinized potato starch, 8-10 g / L of yeast extract powder, 0.3-0.5 g / L of magnesium sulfate heptahydrate, 0.3-0.5 g / L of diammonium hydrogen phosphate, and 1-1.2 g / L of calcium lactate; and the supplementary solution comprises 15-18 g / L of peanut meal extract II, 10-12 g / L of erythritol, 3-5 g / L of potassium chloride, 10-12 g / L of ammonium sulfate, and 5-8 g / L of brown algae polysaccharide.

[0007] The preparation method of peanut meal extract I, peanut meal extract II and peanut meal biochar comprises the following steps:

[0008] Step 1, peanut meal is added with steviol glycoside, lipase and water to prepare homogenate I, the pH is adjusted to 6.5-7.0, Aspergillus oryzae spore suspension and Pichia pastoris seed liquid are inoculated, fermented at 30-35° C. for 36-48 hours, ultrasonicated for 40-60 minutes after the fermentation is completed, centrifuged to obtain precipitate I and supernatant I, and the supernatant I is dried to obtain peanut meal extract I;

[0009] Step 2, adding water to the precipitate I obtained in step 1 to prepare a homogenate II, adjusting the pH to 7.0-7.5, adding hemicellulase, and performing enzymolysis at 35-40° C. for 0.5-1 day; then adjusting the pH to 7.0-7.5, adding Bacillus subtilis protease and pronase, and performing enzymolysis at 35-40° C. for 1-1.5 days. After the enzymolysis is completed, inactivating the enzyme at 80-90° C., centrifuging, and obtaining supernatant II and precipitate II, adjusting the pH of supernatant II to 4.5-5.0 with 1.0 mol / L hydrochloric acid solution, stirring for 30-40 min, and then standing for 20-30 min, then adding 0.5 mol / L sodium chloride solution, stirring for 1-1.5 h, standing for 20-30 min, centrifuging, taking precipitate III, and drying to obtain peanut meal extract II;

[0010] Step 3: Take the precipitate II obtained in step 2, soak it in a potassium permanganate solution with a mass fraction of 0.1-0.15% for 20-24 hours, then dry it, add shell powder, place it in an oxygen-free environment, calcine it at 600-800°C, then soak it in a phosphoric acid solution with a mass fraction of 6-8% for 6-8 hours, and dry it to obtain peanut meal biochar.

[0011] Preferably, in step 1 of the preparation method, the inoculation amount of the Aspergillus oryzae spore suspension is 0.2-0.25% of the volume of the homogenate I, and the inoculation amount of the Pichia pastoris seed solution is 0.1-0.3% of the volume of the homogenate I; the number of spores in the Aspergillus oryzae spore suspension is 1×10 4 ~1×10 5 / mL, and the OD value of Pichia pastoris seed solution is 0.5~0.7.

[0012] Preferably, in step 1 of the above preparation method, the amount of stevioside added is 5-8% of the mass of the peanut meal, and the amount of lipase added is 0.3-0.6% of the mass of the peanut meal; the enzyme activity of the lipase is 20000U / g.

[0013] Preferably, in step 2 of the above preparation method, the amount of hemicellulase added is 0.1-0.15% of the mass of precipitate I; the enzyme activity of the hemicellulase is 20000 U / g.

[0014] Preferably, in step 2 of the above preparation method, the amount of Bacillus subtilisin added is 0.08-0.1% of the mass of precipitate I, and the amount of pronase added is 0.3-0.35% of the mass of precipitate I; the enzyme activity of the Bacillus subtilisin is 150U / mg, and the enzyme activity of the pronase is 7000U / g.

[0015] Preferably, in step 2 of the above preparation method, the volume ratio of supernatant II to sodium chloride solution is 1:(0.6-0.8).

[0016] Preferably, in step 3 of the above preparation method, the amount of shell powder added is 0.2-0.25% of the mass of precipitate II.

[0017] Preferably, the peanut meal used in the above preparation method is low-temperature pressed peanut meal.

[0018] The present invention provides a method for preparing the gelatinized potato starch, which specifically comprises the following steps:

[0019] The potato starch is added with water to be suspended, and then heated at 60-70°C until it becomes transparent, and then cooled to room temperature, dried, and crushed to obtain gelatinized potato starch.

[0020] The present invention provides a specific use of the culture medium, that is, the culture medium is used for producing avermectin by microbial fermentation.

[0021] The present invention provides a method for producing avermectin using the above culture medium, which specifically comprises the following steps:

[0022] Step A, preparing a basic culture solution and a replenishing solution: adding water to peanut meal extract I, peanut meal biochar, gelatinized potato starch, yeast extract powder, magnesium sulfate heptahydrate, diammonium phosphate, and calcium lactate, mixing them evenly, and ultrasonically treating them for 30-40 minutes to prepare a basic culture solution; adding water to peanut meal extract II, erythritol, potassium chloride, ammonium sulfate, and brown algae polysaccharide to prepare a replenishing solution;

[0023] Step B, inoculating the production strain into the basic culture solution obtained in step A, culturing statically for 20-24 hours, then culturing under stirring for 48-72 hours, adding supplementary solution, and culturing under stirring for 144-168 hours to obtain a fermentation solution containing avermectin.

[0024] Preferably, the volume ratio of the basal culture solution to the supplementary solution in the above step B is 1:(0.3-0.5).

[0025] The present invention has the following beneficial effects:

[0026] 1. In the present invention, peanut meal is used as the nutrient component of the culture medium, and the components in the peanut meal are effectively separated, extracted and enriched to different degrees by fermentation, enzymolysis and the like, to obtain peanut meal extract I and peanut meal extract II, and the remaining peanut meal components are used to prepare peanut meal biochar. Peanut meal extract I is used in the basic culture solution, which can effectively promote the reproduction of avermectin-producing bacteria and enhance bacterial vitality; peanut meal biochar is added to the basic culture solution, and the porous structure of biochar is used to effectively load the production bacteria therein, expand its living space, and avoid the influence of metabolites produced in its growth activities on its growth and reproduction; peanut meal extract II is used in the rehydration, which can induce and promote the production bacteria to continuously produce avermectin. By separating and extracting the peanut meal components and using different extracts at different fermentation stages, the uncertainty of the nutrient components in the culture medium caused by the different components of different batches of peanut meal can be effectively avoided, which affects the growth and product synthesis of the production bacteria and causes different avermectin yields in different batches. The fermentation medium prepared using different peanut extracts has relatively stable components and a high and stable avermectin yield.

[0027] 2. In the process of preparing peanut meal extract, the present invention adopts the method of enzymatic hydrolysis and fermentation. The addition of lipase can effectively decompose the fat component in the peanut meal to obtain glycerol and fatty acid components. These components can play the role of defoaming agents to a certain extent. When used in fermentation production, the generation of foam can be effectively avoided; part of shell powder is added in the process of preparing peanut meal biochar. The shell powder will decompose into calcium oxide and carbon dioxide at high temperature. Carbon dioxide helps to etch the peanut meal channel. Calcium oxide is used to prepare the culture medium and forms calcium hydroxide when it meets water, so that the culture medium is weakly alkaline, providing a suitable growth environment for avermectin production bacteria.

[0028] 3. In the present invention, the basic culture solution and the supplementary liquid components in the culture medium are screened to obtain the culture medium components. The basic culture solution can effectively promote the proliferation and growth of the production bacteria and produce some metabolites. The addition of the supplementary liquid can induce and promote the production bacteria to synthesize and accumulate a large amount of avermectin while providing nutrients for the production bacteria. The fermentation production of avermectin using the culture medium can effectively avoid the situation where the yield difference between batches caused by the complexity and uncertainty of the culture medium components is large, and the avermectin yield is large and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 :The variation curve of biomass in fermentation broth at different stages of avermectin production;

[0030] Figure 2 :The Bla titer change curve in fermentation broth at different stages of avermectin production. DETAILED DESCRIPTION

[0031] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, modifications to various equivalent forms of the present invention by those skilled in the art all fall within the scope protected by the claims of this application. The peanut meal used in the following embodiments is low-temperature pressed peanut meal purchased from an edible oil factory; the Aspergillus oryzae spore suspension and Pichia pastoris seed liquid used are common strains purchased from the General Microbiological Center of the China Microbiological Culture Collection Administration and expanded and cultured in the company's laboratory; the enzymes used are all finished enzymes publicly sold by a company in Shanghai, and enzymes with the same enzyme activity purchased from other channels can also achieve similar effects.

[0032] Example 1

[0033] (1) Preparation of peanut meal extract I, peanut meal extract II, and peanut meal biochar:

[0034] Step 1: Dry and crush the peanut meal, add stevioside (5% of the mass of the peanut meal), lipase with an enzyme activity of 20,000 U / g (0.6% of the mass of the peanut meal), and appropriate amount of water to make a homogenate I, and adjust the pH to 7.0; then inoculate 1.8×10 4 A suspension of Aspergillus oryzae spores (inoculation amount is 0.2% of the homogenate volume) with an OD value of 0.7 and a seed solution of Pichia pastoris (inoculation amount is 0.3% of the homogenate volume) were inoculated, and fermented at 35°C for 36 hours. After the fermentation was completed, ultrasonic treatment was performed for 40 minutes, and the mixture was centrifuged to obtain a precipitate I and a supernatant I. The supernatant I was dried to obtain a peanut meal extract I.

[0035] Step 2, add appropriate amount of water to the precipitate I obtained in step 1 to prepare homogenate II, adjust the pH to 7.5, add hemicellulase with an enzyme activity of 20000U / g (the amount added is 0.1% of the mass of precipitate I), and perform enzymolysis at 40°C for 0.5 day; then adjust the pH to 7.5, add subtilisin with an enzyme activity of 150U / mg (the amount added is 0.08% of the mass of precipitate I), pronase with an enzyme activity of 7000U / g (the amount added is 0.1% of the mass of precipitate I), and add 100U / mg lysine protease with an enzyme activity of 100U / mg (the amount added is 0.08% of the mass of precipitate I). The mixture was enzymolyzed at 40℃ for 1 day. After the enzymolysis was completed, the enzyme was inactivated at 90℃ and centrifuged to obtain supernatant II and precipitate II. The pH of supernatant II was adjusted to 5.0 with 1.0mol / L hydrochloric acid solution, stirred for 30min and then allowed to stand for 30min. A 0.5mol / L sodium chloride solution (the amount added was 0.6 times the volume of supernatant II) was added and stirred for 1.5h. The mixture was allowed to stand for 30min and centrifuged to obtain precipitate III, which was dried to obtain peanut meal extract II.

[0036] Step 3: Take the precipitate II obtained in step 2, soak it in a potassium permanganate solution with a mass fraction of 0.15% for 20 hours and then dry it, add shell powder (the amount added is 0.2% of the mass of precipitate II), place it in an oxygen-free environment, calcine it at 800°C, and then soak it in a phosphoric acid solution with a mass fraction of 8% for 6 hours and dry it to obtain peanut meal biochar.

[0037] (2) Preparation of potato gelatinized potato starch:

[0038] The potato starch is added with water to be suspended, and then heated at 70°C until it becomes transparent, and then cooled to room temperature, dried, and crushed to obtain gelatinized potato starch.

[0039] (3) Preparing a culture medium and using the culture medium for avermectin production:

[0040] Step A, preparing a basic culture solution and a replenishing solution: according to 15 g / L peanut meal extract I, 25 g / L peanut meal biochar, 20 g / L gelatinized potato starch, 8 g / L yeast extract powder, 0.5 g / L magnesium sulfate heptahydrate, 0.3 g / L diammonium phosphate, and 1.2 g / L calcium lactate, take each raw material, add water and mix evenly, and ultrasonically treat for 40 minutes to prepare a basic culture solution; according to 18 g / L peanut meal extract II, 10 g / L erythritol, 5 g / L potassium chloride, 10 g / L ammonium sulfate, and 8 g / L brown algae polysaccharide, take the raw materials and add water to prepare a replenishing solution;

[0041] Step B, inoculating Streptomyces avermitilis into the basic culture solution obtained in step A at an inoculation rate of 5%, with a ventilation rate of 3000 L / h, a temperature of 25°C, culturing for 20 hours, and then culturing for 72 hours at a stirring speed of 150 r / min; then adding supplementary liquid according to 0.3 times the volume of the basic culture solution, and continuing to culture for 148 hours at a stirring speed of 150 r / min to obtain a fermentation solution containing avermectin.

[0042] Example 2

[0043] (1) Preparation of peanut meal extract I, peanut meal extract II, and peanut meal biochar:

[0044] Step 1: Dry and crush the peanut meal, add stevioside (8% of the mass of the peanut meal), lipase with an enzyme activity of 20,000 U / g (0.3% of the mass of the peanut meal), and appropriate amount of water to make a homogenate I, and adjust the pH to 6.5; then inoculate spores in an amount of 6.8×10 4A suspension of Aspergillus oryzae spores (inoculation amount is 0.25% of the homogenate volume) with an OD value of 0.5 was inoculated with a seed solution of Pichia pastoris (inoculation amount is 0.1% of the homogenate volume), and the mixture was fermented at 30°C for 48 hours. After the fermentation was completed, the mixture was ultrasonicated for 60 minutes and centrifuged to obtain a precipitate I and a supernatant I. The supernatant I was dried to obtain a peanut meal extract I.

[0045] Step 2, add appropriate amount of water to the precipitate I obtained in step 1 to prepare homogenate II, adjust the pH to 7.0, add hemicellulase with an enzyme activity of 20000U / g (the amount added is 0.15% of the mass of precipitate I), and perform enzymolysis at 35°C for 1 day; then adjust the pH to 7.0, add subtilisin with an enzyme activity of 150U / mg (the amount added is 0.1% of the mass of precipitate I), pronase with an enzyme activity of 7000U / g (the amount added is 0.1% of the mass of precipitate I), and the mixture is stirred for 1 h. The mixture was enzymolyzed at 35°C for 1.5 days. After the enzymolysis was completed, the enzyme was inactivated at 80°C and centrifuged to obtain supernatant II and precipitate II. The pH of supernatant II was adjusted to 4.5 with 1.0 mol / L hydrochloric acid solution, stirred for 40 min, and then allowed to stand for 20 min. A 0.5 mol / L sodium chloride solution (the amount added was 0.8 times the volume of supernatant II) was added and stirred for h. The mixture was allowed to stand for 20 min and centrifuged. The precipitate III was taken and dried to obtain peanut meal extract II.

[0046] Step 3: Take the precipitate II obtained in step 2, soak it in a 0.1% potassium permanganate solution for 24 hours and then dry it, add shell powder (the amount added is 0.25% of the mass of precipitate II), place it in an oxygen-free environment, calcine it at 600°C, and then soak it in a 6% phosphoric acid solution for 8 hours and dry it to obtain peanut meal biochar.

[0047] (2) Preparation of gelatinized potato starch:

[0048] The potato starch is added with water to be suspended, and then heated at 60°C until it becomes transparent, and then cooled to room temperature, dried, and crushed to obtain gelatinized potato starch.

[0049] (3) Preparing a culture medium and using the culture medium for avermectin production:

[0050] Step A, preparing a basic culture solution and a replenishing solution: according to 12 g / L of peanut meal extract I, 20 g / L of peanut meal biochar, 30 g / L of gelatinized potato starch, 10 g / L of yeast extract powder, 0.3 g / L of magnesium sulfate heptahydrate, 0.5 g / L of diammonium phosphate, and 1 g / L of calcium lactate, each raw material is added with water and mixed evenly, and then ultrasonically treated for 30 minutes to prepare a basic culture solution; according to 15 g / L of peanut meal extract II, 12 g / L of erythritol, 3 g / L of potassium chloride, 12 g / L of ammonium sulfate, and 5 g / L of brown algae polysaccharide, the raw materials are added with water to prepare a replenishing solution;

[0051] Step B, inoculating Streptomyces avermitilis into the basic culture solution obtained in step A at an inoculation rate of 5%, with a ventilation rate of 3000 L / h, a temperature of 25°C, culturing for 24 hours, and then culturing for 72 hours at a stirring speed of 150 r / min; then adding a replenishing solution according to 0.5 times the volume of the basic culture solution, and continuing to culture for 144 hours at a stirring speed of 150 r / min to obtain a fermentation solution containing avermectin.

[0052] Example 3

[0053] (1) Preparation of peanut meal extract I, peanut meal extract II, and peanut meal biochar:

[0054] Step 1: Dry and crush the peanut meal, add stevioside (6% of the mass of the peanut meal), lipase with an enzyme activity of 20000U / g (0.5% of the mass of the peanut meal), and appropriate amount of water to make a homogenate I, and adjust the pH to 7.0; then inoculate 0.98×10 5 A suspension of Aspergillus oryzae spores (inoculation amount is 0.2% of the homogenate volume) with an OD value of 0.6 was inoculated with a seed solution of Pichia pastoris (inoculation amount is 0.2% of the homogenate volume), and fermented at 35°C for 40 hours. After the fermentation was completed, ultrasonic treatment was performed for 50 minutes, and centrifugation was performed to obtain a precipitate I and a supernatant I. The supernatant I was dried to obtain a peanut meal extract I.

[0055] Step 2, add appropriate amount of water to the precipitate I obtained in step 1 to prepare homogenate II, adjust the pH to 7.5, add hemicellulase with an enzyme activity of 20000U / g (the amount added is 0.15% of the mass of precipitate I), and perform enzymolysis at 40°C for 1 day; then adjust the pH to 7.5, add subtilisin with an enzyme activity of 150U / mg (the amount added is 0.09% of the mass of precipitate I), pronase with an enzyme activity of 7000U / g (the amount added is 0.35%), enzymolysis at 35℃ for 1.5 days, after the enzymolysis is completed, the enzyme is inactivated at 90℃, centrifuged to obtain supernatant Ⅱ and precipitate Ⅱ, the supernatant Ⅱ is adjusted to pH 4.5 with 1.0mol / L hydrochloric acid solution, stirred for 40min and then allowed to stand for 30min, then 0.5mol / L sodium chloride solution (the amount added is 0.7 times the volume of supernatant Ⅱ) is added and stirred for 1.5h, allowed to stand for 25min, centrifuged, precipitate Ⅲ is taken, and dried to obtain peanut meal extract Ⅱ;

[0056] Step 3: Take the precipitate II obtained in step 2, soak it in a potassium permanganate solution with a mass fraction of 0.13% for 24 hours and then dry it, add shell powder (the amount added is 0.25% of the mass of precipitate II), place it in an oxygen-free environment, calcine it at 700°C, and then soak it in a 7% phosphoric acid solution for 7 hours and dry it to obtain peanut meal biochar.

[0057] (2) Preparation of gelatinized potato starch:

[0058] The potato starch is added with water to be suspended, and then heated at 65°C until it becomes transparent, and then cooled to room temperature, dried, and crushed to obtain gelatinized potato starch.

[0059] (3) Preparing a culture medium and using the culture medium for avermectin production:

[0060] Step A, preparing a basic culture solution and a replenishing solution: according to 13 g / L peanut meal extract I, 22 g / L peanut meal biochar, 25 g / L gelatinized potato starch, 9 g / L yeast extract powder, 0.4 g / L magnesium sulfate heptahydrate, 0.4 g / L diammonium phosphate, and 1 g / L calcium lactate, take each raw material and add water to mix evenly, and ultrasonically treat for 40 minutes to prepare a basic culture solution; according to 17 g / L peanut meal extract II, 11 g / L erythritol, 4 g / L potassium chloride, 11 g / L ammonium sulfate, and 6 g / L fucoidan, take the raw materials and add water to prepare a replenishing solution;

[0061] Step B, inoculating Streptomyces avermitilis into the basic culture solution obtained in step A at an inoculation rate of 5%, with a ventilation rate of 3000 L / h, a temperature of 25°C, culturing for 24 hours, and then culturing for 48 hours at a stirring speed of 150 r / min; then adding supplementary liquid according to 0.4 times the volume of the basic culture solution, and continuing to culture for 168 hours at a stirring speed of 150 r / min to obtain a fermentation solution containing avermectin.

[0062] Comparative Example 1

[0063] Preparation of gelatinized potato starch: Add water to potato starch until it is suspended, then heat it at 65°C until it becomes transparent, then cool it to room temperature, dry it, and grind it to obtain gelatinized potato starch.

[0064] Prepare the replenishing solution: Take the raw materials according to 4g / L potassium chloride, 11g / L ammonium sulfate, and 6g / L brown algae polysaccharide and add water to make the replenishing solution.

[0065] Prepare basic culture medium: according to 30g / L peanut meal, 25g / L gelatinized potato starch, 9g / L yeast extract powder, 0.4g / L magnesium sulfate heptahydrate, 0.4g / L diammonium hydrogen phosphate, and 1g / L calcium lactate, add water and mix well, adjust the pH to 7.0, and obtain the basic culture medium.

[0066] Fermentation production of avermectin: Inoculate Streptomyces avermectin into the above-prepared basic culture solution at an inoculation rate of 5%, with a ventilation rate of 3000L / h, a temperature of 25°C, and culture for 24 hours, followed by culture at a stirring speed of 150r / min for 72 hours; then add supplementary solution at 0.4 times the volume of the basic culture solution, and continue to culture at a stirring speed of 150r / min for 168 hours to obtain a fermentation solution containing avermectin.

[0067] Comparative Example 2

[0068] Preparation of peanut meal extract: Dry and crush the peanut meal, add appropriate amount of water to make homogenate I, adjust the pH to 7.0; then add a composite protease with an enzyme activity of 120U / mg (the added amount is 0.3% of the mass of the peanut meal), hydrolyze at 40°C for 2 days, centrifuge, take the supernatant and dry it to obtain the peanut meal extract.

[0069] Preparation of rehydration solution: 17 g / L peanut meal extract, 11 g / L erythritol, 4 g / L potassium chloride, and 11 g / L ammonium sulfate were added to the raw materials and water to prepare rehydration solution;

[0070] Prepare basic culture medium: according to 25g / L potato starch, 9g / L yeast extract powder, 0.4g / L magnesium sulfate heptahydrate, 0.4g / L diammonium hydrogen phosphate, and 1g / L calcium lactate, add water and mix well, adjust the pH to 7.0, and obtain the basic culture medium.

[0071] Fermentation production of avermectin: Inoculate Streptomyces avermectin into the above-prepared basic culture solution at an inoculation rate of 5%, with a ventilation rate of 3000L / h, a temperature of 25°C, and culture for 24 hours, followed by culture at a stirring speed of 150r / min for 48 hours; then add supplementary solution at 0.4 times the volume of the basic culture solution, and continue to culture at a stirring speed of 150r / min for 168 hours to obtain a fermentation solution containing avermectin.

[0072] Comparative Example 3

[0073] (1) Preparation of peanut meal extract I and peanut meal extract II:

[0074] Step 1: Dry and crush the peanut meal, add stevioside (the amount added is 6% of the mass of the peanut meal) and appropriate amount of water to make a homogenate I, adjust the pH to 7.0, and then inoculate spores with a number of 0.98×10 5 The Aspergillus oryzae spore suspension (inoculation amount is 0.2% of the homogenate volume) was fermented at 35°C for 40 hours. After the fermentation was completed, the mixture was ultrasonicated for 50 minutes and centrifuged to obtain precipitate I and supernatant I. The supernatant I was dried to obtain peanut meal extract I.

[0075] Step 2, adding an appropriate amount of water to the precipitate I obtained in step 1 to prepare a homogenate II, adjusting the pH to 7.5, adding a composite protease with an enzyme activity of 120 U / mg (the amount added is 0.3% of the mass of the precipitate I), performing enzymolysis at 40° C. for 1 day and centrifuging, taking the supernatant and drying it to obtain the peanut meal extract II;

[0076] (2) Preparing a culture medium and using the culture medium for avermectin production:

[0077] Step A, preparing a basic culture solution and a supplement solution: according to 13 g / L peanut meal extract I, 9 g / L yeast extract powder, 0.4 g / L magnesium sulfate heptahydrate, 0.4 g / L diammonium phosphate, and 1 g / L calcium lactate, take each raw material and add water to mix evenly to prepare a basic culture solution; according to 17 g / L peanut meal extract II, 4 g / L potassium chloride, and 11 g / L ammonium sulfate, take the raw material and add water to prepare a supplement solution;

[0078] Step B, inoculating Streptomyces avermitilis into the basic culture solution obtained in step A at an inoculation rate of 5%, with a ventilation rate of 3000 L / h, a temperature of 25°C, culturing for 24 hours, and then culturing for 48 hours at a stirring speed of 150 r / min; then adding supplementary liquid according to 0.4 times the volume of the basic culture solution, and continuing to culture for 168 hours at a stirring speed of 150 r / min to obtain a fermentation solution containing avermectin.

[0079] Performance Testing

[0080] 1. Biomass Determination

[0081] Accurately measure 50 mL of the fermentation broth of Example 1 to Example 3 and Comparative Example 1 to Comparative Example 3, place it in a 250 mL conical flask, add an appropriate amount of filter aid, heat to about 90° C., filter, wash with water, and dry to constant weight, and calculate the biomass according to the following formula.

[0082] Biomass (g / L) = (W2-W0-W1) × 20

[0083] Wherein, W0 is the mass of filter cloth, g; W1 is the mass of filter aid, g; W2 is the mass after drying, g; and 20 is the conversion factor.

[0084] 2. Bla titer determination

[0085] Accurately measure 2 mL of the fermentation broth of Example 1 to Example 3 and Comparative Example 1 to Comparative Example 3 and place it in a 50 mL volumetric flask, add an appropriate amount of methanol, sonicate for 15 min, fix the volume, filter, and perform HPLC analysis according to the chromatographic conditions in the prior art. The injection volume is 20 μL, the peak area is recorded, and the Bla titer is calculated by the external standard method according to the following formula.

[0086] Bla titer (g / L) = (standard concentration × sample peak area × dilution factor) / standard peak area

[0087] 3. Results and Discussion

[0088] since Figure 1 , Figure 2It can be seen that in the fermentation broth of Examples 1 to 3, in the early stage of fermentation, the mycelium grows rapidly and the biomass continues to increase. Later, with the addition of replenishing liquid, the feed liquid is diluted and the biomass decreases, but then grows rapidly. During the entire fermentation cycle, the biomass in the fermentation broth of Examples 1 to 3 is significantly higher than the biomass in the fermentation broth of Comparative Examples 1 to 3. It can be seen that the basic culture solution of the present invention is conducive to the growth and reproduction of Streptomyces avermitilis.

[0089] Abamectin is synthesized by Streptomyces avermitilis after the mycelium grows and reproduces to a certain stage, and the Bla titer continues to increase. After the addition of feed, the Bla titer decreases due to the dilution effect, and then continues to increase. The Bla titer of the fermentation broth of Example 1-Example 3 is higher than that of the fermentation broth of Comparative Example 1-Comparative Example 2. It can be seen that the addition of feed can promote the synthesis of avermectin.

[0090] By observing the fermentation broths, it was found that the fermentation broths of Comparative Examples 1 to 3 contained obvious foams, which would affect the yield of avermectin to a certain extent.

[0091] The fermentation production of avermectin using the culture medium prepared by the present invention can significantly increase the yield.

Claims

1. A fermentation medium, characterized in that It contains a basic culture solution and a supplement solution, wherein the basic culture solution contains 12-15 g / L of peanut meal extract I, 20-25 g / L of peanut meal biochar, 20-30 g / L of gelatinized potato starch, 8-10 g / L of yeast extract powder, 0.3-0.5 g / L of magnesium sulfate heptahydrate, 0.3-0.5 g / L of diammonium hydrogen phosphate, and 1-1.2 g / L of calcium lactate; the supplement solution contains 15-18 g / L of peanut meal extract II, 10-12 g / L of erythritol, 3-5 g / L of potassium chloride, 10-12 g / L of ammonium sulfate, and 5-8 g / L of fucoidan; The preparation method of peanut meal extract I, peanut meal extract II and peanut meal biochar comprises the following steps: Step 1, peanut meal is added with steviol glycoside, lipase and water to prepare homogenate I, the pH is adjusted to 6.5-7.0, Aspergillus oryzae spore suspension and Pichia pastoris seed liquid are inoculated, fermented at 30-35° C. for 36-48 hours, ultrasonicated for 40-60 minutes after the fermentation is completed, centrifuged to obtain precipitate I and supernatant I, and the supernatant I is dried to obtain peanut meal extract I; Step 2, adding water to the precipitate I obtained in step 1 to prepare a homogenate II, adjusting the pH to 7.0-7.5, adding hemicellulase, and performing enzymolysis at 35-40° C. for 0.5-1 day; then adjusting the pH to 7.0-7.5, adding Bacillus subtilis protease and pronase, and performing enzymolysis at 35-40° C. for 1-1.5 days. After the enzymolysis is completed, inactivating the enzyme at 80-90° C., centrifuging, and obtaining supernatant II and precipitate II, adjusting the pH of supernatant II to 4.5-5.0 with 1.0 mol / L hydrochloric acid solution, stirring for 30-40 min, and then standing for 20-30 min, then adding 0.5 mol / L sodium chloride solution, stirring for 1-1.5 h, standing for 20-30 min, centrifuging, taking precipitate III, and drying to obtain peanut meal extract II; Step 3: Take the precipitate II obtained in step 2, soak it in a potassium permanganate solution with a mass fraction of 0.1-0.15% for 20-24 hours, then dry it, add shell powder, place it in an oxygen-free environment, calcine it at 600-800°C, then soak it in a phosphoric acid solution with a mass fraction of 6-8% for 6-8 hours, and dry it to obtain peanut meal biochar.

2. The fermentation medium according to claim 1, characterized in that In step 1, the inoculation amount of the Aspergillus oryzae spore suspension is 0.2-0.25% of the volume of the homogenate I, and the inoculation amount of the Pichia pastoris seed solution is 0.1-0.3% of the volume of the homogenate I; the number of spores in the Aspergillus oryzae spore suspension is 1×10 4 ~1×10 5 / mL, and the OD value of Pichia pastoris seed solution is 0.5~0.

7.

3. The fermentation medium according to claim 1, characterized in that In the step 1, the amount of stevioside added is 5-8% of the mass of the peanut meal, and the amount of lipase added is 0.3-0.6% of the mass of the peanut meal; the enzyme activity of the lipase is 20000U / g.

4. The fermentation medium according to claim 1, characterized in that In the step 2, the amount of hemicellulase added is 0.1-0.15% of the mass of precipitate I; the enzyme activity of the hemicellulase is 20000 U / g.

5. The fermentation medium according to claim 1, characterized in that In step 2, the amount of subtilisin added is 0.08-0.1% of the mass of precipitate I, and the amount of pronase added is 0.3-0.35% of the mass of precipitate I; the enzyme activity of subtilisin is 150U / mg, and the enzyme activity of pronase is 7000U / g.

6. The fermentation medium according to claim 1, characterized in that In step 2, the volume ratio of supernatant II to sodium chloride solution is 1:(0.6-0.8).

7. The fermentation medium according to claim 1, characterized in that In step 3, the amount of shell powder added is 0.2-0.25% of the mass of precipitation II.

8. The fermentation medium according to claim 1, characterized in that The preparation method of the gelatinized potato starch comprises the following steps: The potato starch is added with water to be suspended, and then heated at 60-70°C until it becomes transparent, and then cooled to room temperature, dried, and crushed to obtain gelatinized potato starch.

9. A method for producing avermectin using the fermentation medium according to any one of claims 1 to 8, characterized in that: The method comprises the following steps: Step A, preparing a basic culture solution and a replenishing solution: adding water to peanut meal extract I, peanut meal biochar, gelatinized potato starch, yeast extract powder, magnesium sulfate heptahydrate, diammonium phosphate, and calcium lactate, mixing them evenly, and ultrasonically treating them for 30-40 minutes to prepare a basic culture solution; adding water to peanut meal extract II, erythritol, potassium chloride, ammonium sulfate, and brown algae polysaccharide to prepare a replenishing solution; Step B, inoculating the production strain into the basic culture solution obtained in step A, culturing statically for 20-24 hours, then culturing under stirring for 48-72 hours, adding supplementary solution, and culturing under stirring for 144-168 hours to obtain a fermentation solution containing avermectin.

10. The method according to claim 9, characterized in that The volume ratio of basal culture medium to supplementary fluid in step B is 1:(0.3-0.5).

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