CN118652830B - A fermentation medium capable of increasing the yield of avermectin - Google Patents
A fermentation medium capable of increasing the yield of avermectin Download PDFInfo
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- CN118652830B CN118652830B CN202411102845.1A CN202411102845A CN118652830B CN 118652830 B CN118652830 B CN 118652830B CN 202411102845 A CN202411102845 A CN 202411102845A CN 118652830 B CN118652830 B CN 118652830B
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Abstract
The invention relates to a fermentation medium capable of improving avermectin yield, and belongs to the technical field of microbial fermentation media. The fermentation medium comprises a basic culture solution and a fluid replacement, wherein the basic culture solution comprises 12-15g/L of peanut meal extract I, 20-25g/L of peanut meal biochar, 20-30g/L of gelatinized potato starch, 8-10g/L of yeast extract powder, 0.3-0.5g/L of magnesium sulfate heptahydrate, 0.3-0.5g/L of diammonium hydrogen phosphate and 1-1.2g/L of calcium lactate, and the fluid replacement comprises 15-18g/L of peanut meal extract II, 10-12g/L of erythritol, 3-5g/L of potassium chloride, 10-12g/L of ammonium sulfate and 5-8g/L of brown algae polysaccharide. When the culture medium provided by the invention is used for fermenting and producing the avermectin, the yield of the avermectin can be obviously improved, and the avermectin has certain stability.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation media, and relates to a fermentation medium capable of improving the yield of abamectin.
Background
Avermectin is a widely used agricultural or veterinary bactericidal, insecticidal, acaricidal agent, also known as amectin. Avermectin is a hexadecanoid macrolide compound with bactericidal, insecticidal, acaricidal and nematicidal activities, which is first developed by university of North China, tochu Korea, etc. and Merck company in the United states, and is produced by fermentation of Streptomyces avermitilis in Streptomyces.
The fermentation medium is an important platform for industrialization of fermentation products, the yield and cost of the avermectin can be directly influenced, the types of the medium used for industrial fermentation are various, the components in the medium, especially the natural components, have a large influence on the fermentation yield, and due to the growth characteristics of the streptomyces avermitilis, batch fermentation can only be used as a main production mode, so that the yield of the avermectin in different fermentation tanks can be greatly different due to strain degradation, different delay periods, carbon nitrogen source feeding demand change and the like, the extraction and refining of the avermectin are limited to a certain extent by the difference, and the production cost is increased, so that it is important to select a better medium.
Disclosure of Invention
In order to improve the yield of the avermectin, the invention provides the culture medium which can effectively improve the fermentation efficiency, reduce the cost consumption and increase the yield of the avermectin, and the culture medium can realize the stable and efficient production of the avermectin.
The invention adopts the following technical scheme to realize the purposes:
A fermentation medium comprises a basic culture solution and a fluid replacement, wherein the basic culture solution comprises 12-15g/L of peanut meal extract I, 20-25g/L of peanut meal biochar, 20-30g/L of gelatinized potato starch, 8-10g/L of yeast extract, 0.3-0.5g/L of magnesium sulfate heptahydrate, 0.3-0.5g/L of diammonium phosphate and 1-1.2g/L of calcium lactate, and the fluid replacement comprises 15-18g/L of peanut meal extract II, 10-12g/L of erythritol, 3-5g/L of potassium chloride, 10-12g/L of ammonium sulfate and 5-8g/L of fucoidin.
The preparation method of the peanut meal extract I, the peanut meal extract II and the peanut meal biochar comprises the following steps:
Step 1, adding stevioside, lipase and water into peanut meal to prepare homogenate I, regulating the pH to 6.5-7.0, inoculating aspergillus oryzae spore suspension and pichia pastoris seed liquid, fermenting for 36-48 hours at the temperature of 30-35 ℃, carrying out ultrasonic treatment for 40-60min after fermentation, centrifuging to obtain sediment I and supernatant I, and drying the supernatant I to obtain a peanut meal extract I;
Step 2, adding water into the sediment I obtained in the step 1 to prepare homogenate II, regulating the pH to 7.0-7.5, adding hemicellulase, carrying out enzymolysis for 0.5-1 day at 35-40 ℃, then regulating the pH to 7.0-7.5, adding subtilisin and pronase, carrying out enzymolysis for 1-1.5 days at 35-40 ℃, inactivating enzyme at 80-90 ℃ after the enzymolysis is finished, centrifuging to obtain a supernatant II and sediment II, regulating the pH of the supernatant II to 4.5-5.0 by using 1.0mol/L hydrochloric acid solution, stirring for 30-40min, then standing for 20-30min, adding 0.5mol/L sodium chloride solution, stirring for 1-1.5h, standing for 20-30min, centrifuging, taking sediment III, and drying to obtain a peanut meal extract II;
And 3, taking the precipitate II obtained in the step 2, soaking the precipitate II in a potassium permanganate solution with the mass fraction of 0.1-0.15% for 20-24 hours, drying, adding shell powder, calcining at 600-800 ℃ in an oxygen-free environment, soaking in a phosphoric acid solution with the mass fraction of 6-8% for 6-8 hours, and drying to obtain the peanut meal biochar.
Preferably, in the 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, the inoculation amount of the pichia pastoris seed liquid is 0.1-0.3% of the volume of the homogenate I, the number of spores in the aspergillus oryzae spore suspension is 1X 10 4~1×105/mL, and the OD value of the pichia pastoris seed liquid is 0.5-0.7.
Preferably, in the step 1 of the preparation method, the addition amount of stevioside is 5-8% of the mass of peanut meal, the addition amount of lipase is 0.3-0.6% of the mass of peanut meal, and the enzyme activity of the lipase is 20000U/g.
Preferably, in the step 2 of the preparation method, the adding amount of the hemicellulase is 0.1-0.15% of the mass of the sediment I, and the enzyme activity of the hemicellulase is 20000U/g.
Preferably, in the step 2 of the preparation method, the addition amount of the subtilisin is 0.08-0.1% of the mass of the sediment I, the addition amount of the pronase is 0.3-0.35% of the mass of the sediment I, the enzyme activity of the subtilisin is 150U/mg, and the enzyme activity of the pronase is 7000U/g.
Preferably, in the step 2 of the preparation method, the volume ratio of the supernatant II to the sodium chloride solution is 1 (0.6-0.8).
Preferably, in the step 3 of the preparation method, the addition amount of the shell powder is 0.2 to 0.25 percent of the mass of the sediment II.
Preferably, the peanut meal used in the preparation method is low-temperature pressed peanut meal.
The invention provides a preparation method of gelatinized potato starch, which specifically comprises the following steps:
adding water into potato starch to obtain suspension, heating at 60-70deg.C to transparent, cooling to room temperature, drying, and pulverizing to obtain gelatinized potato starch.
The invention provides a specific application of the culture medium, namely the culture medium is used for producing abamectin by microbial fermentation.
The invention provides a method for producing abamectin by utilizing the culture medium, which specifically comprises the following steps:
step A, preparing a basic culture solution and a fluid replacement, namely uniformly mixing peanut meal extract I, peanut meal biochar, gelatinized potato starch, yeast extract powder, magnesium sulfate heptahydrate, diammonium phosphate and calcium lactate with water, and performing ultrasonic treatment for 30-40min to prepare the basic culture solution;
and B, inoculating a production strain into the basic culture solution obtained in the step A, standing and culturing for 20-24 hours, culturing for 48-72 hours under a stirring state, adding a fluid replacement, and culturing for 144-168 hours under the stirring state to obtain the avermectin-containing fermentation liquor.
Preferably, the volume ratio of the basic culture solution to the fluid replacement in the step B is 1 (0.3-0.5).
The invention has the following beneficial effects:
1. According to the invention, peanut meal is used as a nutrient component of a culture medium, components in the peanut meal are effectively separated, extracted and enriched to different degrees in a fermentation mode, an enzymolysis mode and the like, so that a peanut meal extract I and a peanut meal extract II are obtained, residual peanut meal components are utilized to prepare peanut meal biochar, the peanut meal extract I is used in a basic culture solution, the propagation of abamectin production bacteria can be effectively promoted, the activity of the bacteria is enhanced, the peanut meal biochar is added into the basic culture solution, the porous structure of the biochar is utilized, the effective load of the production bacteria can be realized, the living space of the production bacteria is enlarged, the influence of metabolites generated in the growth activity of the production bacteria on the growth and propagation of the production bacteria can be avoided, and the peanut meal extract II is used in a fluid replacement, and the continuous production of abamectin by the production bacteria can be induced and promoted. By separating and extracting peanut meal components and using different extracts in different fermentation stages, uncertainty of nutritional components in a culture medium caused by different peanut meal components in different batches can be effectively avoided, growth of production bacteria and synthesis of products are affected, and different avermectin yields in different batches are caused. The fermentation culture medium prepared by using different peanut extracts has relatively stable components and higher and stable avermectin yield.
2. The preparation method adopts an enzymolysis and fermentation mode in the preparation process of the peanut meal extract, the lipase can be added to effectively decompose fat components in the peanut meal to obtain glycerol and fatty acid components, the components can play a role of a defoaming agent to a certain extent, foam can be effectively avoided when the peanut meal extract is used for fermentation production, part of shell powder is added in the preparation process of the peanut meal biochar, the shell powder can be decomposed into calcium oxide and carbon dioxide at high temperature, the carbon dioxide is helpful for etching peanut meal channels, and the calcium oxide is used for preparing a culture medium to form calcium hydroxide when meeting water, so that the culture medium presents weak alkalinity, and a proper growth environment is provided for avermectin production bacteria.
3. According to the invention, the basic culture solution and the fluid replacement 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 to generate partial metabolites, and the addition of the fluid replacement can induce and promote the mass synthesis and accumulation of abamectin by the production bacteria while providing nutrition components for the production bacteria. The adoption of the culture medium for the fermentation production of the avermectin can effectively avoid the condition that the yield difference between batches is large due to the complex components and uncertainty of the culture medium, and the yield of the avermectin is large and stable.
Drawings
FIG. 1 shows the variation curves of biomass in fermentation broth at different stages of avermectin production;
FIG. 2 shows Bla titer curves in fermentation broths at different stages of avermectin production.
Detailed Description
The present application is further illustrated below with reference to specific examples, which are to be construed as merely illustrative of the application and not limiting of its scope, as various equivalent modifications to the application will fall within the scope of the claims after reading the 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 the pichia pastoris seed liquid are obtained by performing expansion culture on common strains purchased from a common microorganism center of China general microbiological culture Collection center in a laboratory of the company, the enzymes used are finished enzymes which are sold by a company in Shanghai, and similar effects can be achieved by using enzymes with the same enzyme activity purchased from other channels.
Example 1
(1) Preparing peanut meal extract I, peanut meal extract II and peanut meal biochar:
Step 1, taking peanut meal, drying, crushing, adding stevioside (the addition amount is 5% of the mass of the peanut meal), enzyme activity is 20000U/g lipase (the addition amount is 0.6% of the mass of the peanut meal), preparing homogenate I by using a proper amount of water, regulating pH to 7.0, inoculating Aspergillus oryzae spore suspension (the inoculation amount is 0.2% of the homogenate volume) with the spore amount of 1.8X10. 10 4/mL, inoculating Pichia pastoris seed solution (the inoculation amount is 0.3% of the homogenate volume) with the OD value of 0.7, fermenting for 36h at 35 ℃, carrying out ultrasonic treatment for 40min after fermentation, centrifuging to obtain sediment I and supernatant I, and drying the supernatant I to obtain a peanut meal extract I;
Step 2, adding a proper amount of water into the sediment I obtained in the step 1 to prepare homogenate II, regulating the pH to 7.5, adding hemicellulase with the enzyme activity of 20000U/g (the adding amount is 0.1% of the mass of the sediment I), carrying out enzymolysis for 0.5 days at 40 ℃, regulating the pH to 7.5, adding subtilisin with the enzyme activity of 150U/mg (the adding amount is 0.08% of the mass of the sediment I), adding pronase with the enzyme activity of 7000U/g (the adding amount is 0.35% of the mass of the sediment I), carrying out enzymolysis for 1 day at 40 ℃, inactivating enzyme at 90 ℃ after the enzymolysis is completed, centrifuging to obtain supernatant II and sediment II, regulating the pH of the supernatant II to 5.0 by using hydrochloric acid solution with the concentration of 1.0mol/L, stirring for 30min, adding sodium chloride solution with the concentration of 0.5mol/L (the adding amount is 0.6 times of the volume of the supernatant II), stirring for 1.5h, standing for 30min, centrifuging, taking the sediment III, and drying to obtain peanut meal extract II;
And 3, taking the precipitate II obtained in the step 2, soaking the precipitate II in a potassium permanganate solution with the mass fraction of 0.15% for 20 hours, drying, adding shell powder (the addition amount is 0.2% of the mass of the precipitate II), calcining at 800 ℃ in an oxygen-free environment, soaking in a phosphoric acid solution with the mass fraction of 8% for 6 hours, and drying to obtain the peanut meal biochar.
(2) Preparing potato gelatinized potato starch:
Adding water into potato starch to form suspension, heating at 70deg.C to transparent, cooling to room temperature, drying, and pulverizing to obtain gelatinized potato starch.
(3) Preparing a culture medium and utilizing the culture medium to produce abamectin:
Step A, preparing a basic culture solution and replenishing the basic culture solution, wherein the basic culture solution is prepared by adding water into raw materials according to 15g/L of peanut meal extract I, 25g/L of peanut meal biochar, 20g/L of gelatinized potato starch, 8g/L of yeast extract powder, 0.5g/L of magnesium sulfate heptahydrate, 0.3g/L of diammonium phosphate and 1.2g/L of calcium lactate, uniformly mixing the raw materials, and performing ultrasonic treatment for 40min, and the replenishing solution is prepared by adding water into the raw materials according to 18g/L of peanut meal extract II, 10g/L of erythritol, 5g/L of potassium chloride, 10g/L of ammonium sulfate and 8g/L of fucoidan;
and B, inoculating streptomyces avermitilis into the basic culture solution obtained in the step A according to the inoculation amount of 5%, wherein the ventilation amount is 3000L/h, the temperature is 25 ℃, standing and culturing for 20h, then culturing for 72h at the stirring speed of 150r/min, adding a fluid replacement according to the volume of 0.3 times of the basic culture solution, and continuously culturing for 148h at the stirring speed of 150r/min to obtain the fermentation liquor containing the avermectin.
Example 2
(1) Preparing peanut meal extract I, peanut meal extract II and peanut meal biochar:
Step 1, taking peanut meal, drying, crushing, adding stevioside (the addition amount is 8% of the mass of the peanut meal), enzyme activity is 20000U/g lipase (the addition amount is 0.3% of the mass of the peanut meal), preparing homogenate I by using a proper amount of water, regulating pH to 6.5, inoculating Aspergillus oryzae spore suspension (the inoculation amount is 0.25% of the homogenate volume) with the spore amount of 6.8X10. 10 4/mL, inoculating Pichia pastoris seed solution (the inoculation amount is 0.1% of the homogenate volume) with the OD value of 0.5, fermenting for 48 hours at 30 ℃, carrying out ultrasonic treatment for 60min after fermentation, centrifuging to obtain sediment I and supernatant I, and drying the supernatant I to obtain a peanut meal extract I;
Step 2, adding a proper amount of water into the sediment I obtained in the step 1 to prepare homogenate II, regulating the pH to 7.0, adding hemicellulase with the enzyme activity of 20000U/g (the adding amount is 0.15% of the mass of the sediment I), carrying out enzymolysis for 1 day at 35 ℃, regulating the pH to 7.0, adding subtilisin with the enzyme activity of 150U/mg (the adding amount is 0.1% of the mass of the sediment I), carrying out enzymolysis for 1.5 days at 35 ℃, inactivating enzyme at 80 ℃ after the enzymolysis is finished, centrifuging to obtain supernatant II and sediment II, regulating the pH of the supernatant II to 4.5 by using hydrochloric acid solution with the concentration of 1.0mol/L, stirring for 40min, then standing for 20min, adding sodium chloride solution with the concentration of 0.5mol/L (the adding amount is 0.8 times of the volume of the supernatant II), stirring for 20min, taking the sediment III, and drying to obtain peanut meal II;
And 3, taking the precipitate II obtained in the step 2, soaking in a potassium permanganate solution with the mass fraction of 0.1% for 24 hours, drying, adding shell powder (the addition amount is 0.25% of the mass of the precipitate II), calcining at 600 ℃ in an oxygen-free environment, soaking in a phosphoric acid solution with the mass fraction of 6% for 8 hours, and drying to obtain the peanut meal biochar.
(2) Preparing gelatinized potato starch:
adding water into potato starch to form suspension, heating at 60deg.C to transparent, cooling to room temperature, drying, and pulverizing to obtain gelatinized potato starch.
(3) Preparing a culture medium and utilizing the culture medium to produce abamectin:
Step A, preparing a basic culture solution and a fluid replacement, namely adding water into each raw material according to 12g/L of peanut meal extract I, 20g/L of peanut meal biochar, 30g/L of gelatinized potato starch, 10g/L of yeast extract powder, 0.3g/L of magnesium sulfate heptahydrate, 0.5g/L of diammonium phosphate and 1g/L of calcium lactate, uniformly mixing the raw materials, and performing ultrasonic treatment for 30min to prepare the basic culture solution, wherein the basic culture solution is prepared according to 15g/L of peanut meal extract II, 12g/L of erythritol, 3g/L of potassium chloride, 12g/L of ammonium sulfate and 5g/L of brown algae polysaccharide;
And B, inoculating streptomyces avermitilis into the basic culture solution obtained in the step A according to the inoculation amount of 5%, wherein the ventilation amount is 3000L/h, the temperature is 25 ℃, standing and culturing for 24h, then culturing for 72h at the stirring speed of 150r/min, adding a fluid replacement according to the volume of 0.5 times of the basic culture solution, and continuously culturing for 144h at the stirring speed of 150r/min to obtain the fermentation liquor containing the avermectin.
Example 3
(1) Preparing peanut meal extract I, peanut meal extract II and peanut meal biochar:
Step 1, taking peanut meal, drying, crushing, adding stevioside (the addition amount is 6% of the mass of the peanut meal), enzyme activity is 20000U/g lipase (the addition amount is 0.5% of the mass of the peanut meal), preparing homogenate I by using a proper amount of water, regulating pH to 7.0, inoculating Aspergillus oryzae spore suspension (the inoculation amount is 0.2% of the homogenate volume) with the spore amount of 0.98X10. 10 5/mL, inoculating Pichia pastoris seed solution (the inoculation amount is 0.2% of the homogenate volume) with the OD value of 0.6, fermenting for 40 hours at 35 ℃, carrying out ultrasonic treatment for 50min after fermentation, centrifuging to obtain sediment I and supernatant I, and drying the supernatant I to obtain a peanut meal extract I;
Step 2, adding a proper amount of water into the sediment I obtained in the step 1 to prepare homogenate II, regulating the pH to 7.5, adding hemicellulase with the enzyme activity of 20000U/g (the adding amount is 0.15% of the mass of the sediment I), carrying out enzymolysis for 1 day at 40 ℃, regulating the pH to 7.5, adding subtilisin with the enzyme activity of 150U/mg (the adding amount is 0.09% of the mass of the sediment I), carrying out enzymolysis for 1.5 days at 35 ℃, inactivating enzyme at 90 ℃ after the enzymolysis is finished, centrifuging to obtain supernatant II and sediment II, regulating the pH of the supernatant II to 4.5 by using hydrochloric acid solution with the concentration of 1.0mol/L, stirring for 40min, then standing for 30min, adding sodium chloride solution with the concentration of 0.5mol/L (the adding amount is 0.7 times of the volume of the supernatant II), stirring for 1.5h, standing for 25min, centrifuging, taking the sediment III, and drying to obtain peanut meal extract II;
and 3, taking the precipitate II obtained in the step 2, soaking the precipitate II in a potassium permanganate solution with the mass fraction of 0.13% for 24 hours, drying, adding shell powder (the addition amount is 0.25% of the mass of the precipitate II), calcining at 700 ℃ in an oxygen-free environment, soaking in a phosphoric acid solution with the mass fraction of 7% for 7 hours, and drying to obtain the peanut meal biochar.
(2) Preparing gelatinized potato starch:
adding water into potato starch to form suspension, heating at 65deg.C to transparent, cooling to room temperature, drying, and pulverizing to obtain gelatinized potato starch.
(3) Preparing a culture medium and utilizing the culture medium to produce abamectin:
Step A, preparing a basic culture solution and a fluid replacement, namely adding water into each raw material according to 13g/L of peanut meal extract I, 22g/L of peanut meal biochar, 25g/L of gelatinized potato starch, 9g/L of yeast extract powder, 0.4g/L of magnesium sulfate heptahydrate, 0.4g/L of diammonium phosphate and 1g/L of calcium lactate, uniformly mixing the raw materials, and performing ultrasonic treatment for 40min to prepare the basic culture solution, wherein the basic culture solution is prepared according to 17g/L of peanut meal extract II, 11g/L of erythritol, 4g/L of potassium chloride, 11g/L of ammonium sulfate and 6g/L of brown algae polysaccharide;
And B, inoculating streptomyces avermitilis into the basic culture solution obtained in the step A according to the inoculation amount of 5%, wherein the ventilation amount is 3000L/h, the temperature is 25 ℃, standing and culturing for 24h, then culturing for 48h at the stirring speed of 150r/min, adding a fluid replacement according to the volume of 0.4 times of the basic culture solution, and continuously culturing for 168h at the stirring speed of 150r/min to obtain the fermentation liquor containing the avermectin.
Comparative example 1
The gelatinized potato starch is prepared by adding water into potato starch to form suspension, heating to transparent state at 65deg.C, cooling to room temperature, drying, and pulverizing.
The preparation of the rehydration liquid is to take the raw materials according to 4g/L of potassium chloride, 11g/L of ammonium sulfate and 6g/L of fucoidin and add water to prepare the rehydration liquid.
Preparing a basic culture solution, namely taking and adding water into raw materials according to 30g/L of peanut meal powder, 25g/L of gelatinized potato starch, 9g/L of yeast extract powder, 0.4g/L of magnesium sulfate heptahydrate, 0.4g/L of diammonium hydrogen phosphate and 1g/L of calcium lactate, uniformly mixing, and regulating the pH value to 7.0 to obtain the basic culture solution.
Fermenting to produce avermectin, inoculating streptomyces avermitilis into the prepared basic culture solution according to the inoculum size of 5%, standing and culturing for 72 hours at the stirring speed of 150r/min after the aeration rate is 3000L/h and the temperature is 25 ℃, adding a fluid replacement according to the volume of 0.4 times of the basic culture solution, and continuously culturing for 168 hours at the stirring speed of 150r/min to obtain the avermectin-containing fermentation liquor.
Comparative example 2
The preparation of the peanut meal extract comprises the steps of taking peanut meal, drying, crushing, preparing a proper amount of water into homogenate I, regulating the pH value to 7.0, adding compound protease with the enzyme activity of 120U/mg (the addition amount is 0.3% of the mass of the peanut meal), carrying out enzymolysis for 2 days at 40 ℃, centrifuging, taking supernatant and drying to obtain the peanut meal extract.
Preparing a rehydration liquid by adding water into the raw materials according to 17g/L of peanut meal extract, 11g/L of erythritol, 4g/L of potassium chloride and 11g/L of ammonium sulfate;
Preparing a basic culture solution, namely taking raw materials according to 25g/L of potato starch, 9g/L of yeast extract powder, 0.4g/L of magnesium sulfate heptahydrate, 0.4g/L of diammonium phosphate and 1g/L of calcium lactate, adding water, uniformly mixing, and regulating the pH value to 7.0 to obtain the basic culture solution.
Fermenting to produce avermectin, inoculating streptomyces avermitilis into the prepared basic culture solution according to the inoculum size of 5%, standing and culturing for 48 hours at the stirring speed of 150r/min after the aeration rate is 3000L/h and the temperature is 25 ℃, adding a fluid replacement according to the volume of 0.4 times of the basic culture solution, and continuously culturing for 168 hours at the stirring speed of 150r/min to obtain the avermectin-containing fermentation liquor.
Comparative example 3
(1) Preparing a peanut meal extract I and a peanut meal extract II:
Step 1, taking peanut meal, drying, crushing, adding stevioside (the addition amount is 6% of the mass of the peanut meal) and a proper amount of water to prepare homogenate I, regulating the pH to 7.0, inoculating Aspergillus oryzae spore suspension (the inoculation amount is 0.2% of the homogenate volume) with the spore amount of 0.98X10 5/mL, fermenting for 40h at 35 ℃, carrying out ultrasonic treatment for 50min after fermentation, centrifuging to obtain sediment I and supernatant I, and drying the supernatant I to obtain a peanut meal extract I;
Step 2, adding a proper amount of water into the precipitate I obtained in the step 1 to prepare homogenate II, regulating the pH to 7.5, adding compound protease with the enzyme activity of 120U/mg (the addition amount is 0.3% of the mass of the precipitate I), carrying out enzymolysis for 1 day at 40 ℃, centrifuging, taking supernatant, and drying to obtain peanut meal extract II;
(2) Preparing a culture medium and utilizing the culture medium to produce abamectin:
Step A, preparing a basic culture solution and a fluid replacement, namely adding water into each raw material according to 13g/L of peanut meal extract I, 9g/L of yeast extract powder, 0.4g/L of magnesium sulfate heptahydrate, 0.4g/L of diammonium phosphate and 1g/L of calcium lactate to uniformly mix to prepare the basic culture solution, and adding water into the raw materials according to 17g/L of peanut meal extract II, 4g/L of potassium chloride and 11g/L of ammonium sulfate to prepare the fluid replacement;
And B, inoculating streptomyces avermitilis into the basic culture solution obtained in the step A according to the inoculation amount of 5%, wherein the ventilation amount is 3000L/h, the temperature is 25 ℃, standing and culturing for 24h, then culturing for 48h at the stirring speed of 150r/min, adding a fluid replacement according to the volume of 0.4 times of the basic culture solution, and continuously culturing for 168h at the stirring speed of 150r/min to obtain the fermentation liquor containing the avermectin.
Performance testing
1. Biomass determination
50ML of the fermentation solutions of example 1-example 3 and comparative example 1-comparative example 3 are accurately measured, placed in a 250mL triangular flask, a proper amount of filter aid is added, heated to about 90 ℃, suction filtered, washed with water, dried to constant weight, and biomass is calculated according to the following formula.
Biomass (g/L) = (W 2-W0-W1) ×20
Wherein W 0 is the mass of filter cloth, g, W 1 is the mass of filter aid, g, W 2 is the mass after drying, and 20 is the conversion coefficient.
Bla potency assay
Accurately measuring 2mL of fermentation liquor of the embodiment 1-the embodiment 3 and the comparative embodiment 1-the comparative embodiment 3, placing the fermentation liquor in a 50mL volumetric flask, adding a proper amount of methanol, carrying out ultrasonic treatment for 15min, fixing the volume, filtering, carrying out HPLC analysis by referring to chromatographic conditions in the prior art, recording peak areas with the sample injection amount of 20 mu L, and calculating Bla titer according to the following formula by adopting an external standard method.
Bla potency (g/L) = (standard concentration x sample peak area x dilution factor)/standard peak area
3. Results and discussion
As can be seen from FIGS. 1 and 2, in the fermentation broths of examples 1-3, hypha grows rapidly and biomass increases continuously in the early fermentation period, then the feed liquid is diluted with the addition of the fluid replacement, and the biomass decreases, but grows rapidly thereafter, and the biomass in the fermentation broths of examples 1-3 is obviously higher than that in the fermentation broths of comparative examples 1-3 in the whole fermentation period, so that the basal culture broth of the invention is beneficial to the growth and propagation of the Streptomyces avermitilis.
The avermectin is synthesized after the growth and propagation of the hyphae of the streptomyces avermitilis to a certain stage, and the Bla titer is continuously increased, after the feeding, the dilution effect Bla titer is reduced to a certain extent, and then the dilution effect Bla titer is continuously increased, and the fermentation liquor Bla titers of the embodiment 1-embodiment 3 are higher than those of the comparison 1-comparison 2, so that the addition of the feeding can promote the synthesis of the avermectin.
By observing the individual broths, the broths of comparative example 1-comparative example 3 had significant foam which affected the avermectin production to some extent.
The culture medium prepared by the invention is used for fermenting and producing the avermectin, and the yield of the avermectin can be obviously improved.
Claims (10)
1. A fermentation culture medium is characterized by comprising a basic culture solution and a fluid replacement, wherein the basic culture solution comprises 12-15g/L of peanut meal extract I, 20-25g/L of peanut meal biochar, 20-30g/L of gelatinized potato starch, 8-10g/L of yeast extract powder, 0.3-0.5g/L of magnesium sulfate heptahydrate, 0.3-0.5g/L of diammonium phosphate and 1-1.2g/L of calcium lactate, and the fluid replacement comprises 15-18g/L of peanut meal extract II, 10-12g/L of erythritol, 3-5g/L of potassium chloride, 10-12g/L of ammonium sulfate and 5-8g/L of brown algae polysaccharide;
The preparation method of the peanut meal extract I, the peanut meal extract II and the peanut meal biochar comprises the following steps:
Step 1, adding stevioside, lipase and water into peanut meal to prepare homogenate I, regulating the pH to 6.5-7.0, inoculating aspergillus oryzae spore suspension and pichia pastoris seed liquid, fermenting for 36-48 hours at the temperature of 30-35 ℃, carrying out ultrasonic treatment for 40-60min after fermentation, centrifuging to obtain sediment I and supernatant I, and drying the supernatant I to obtain a peanut meal extract I;
Step 2, adding water into the sediment I obtained in the step 1 to prepare homogenate II, regulating the pH to 7.0-7.5, adding hemicellulase, carrying out enzymolysis for 0.5-1 day at 35-40 ℃, then regulating the pH to 7.0-7.5, adding subtilisin and pronase, carrying out enzymolysis for 1-1.5 days at 35-40 ℃, inactivating enzyme at 80-90 ℃ after the enzymolysis is finished, centrifuging to obtain a supernatant II and sediment II, regulating the pH of the supernatant II to 4.5-5.0 by using 1.0mol/L hydrochloric acid solution, stirring for 30-40min, then standing for 20-30min, adding 0.5mol/L sodium chloride solution, stirring for 1-1.5h, standing for 20-30min, centrifuging, taking sediment III, and drying to obtain a peanut meal extract II;
And 3, taking the precipitate II obtained in the step 2, soaking the precipitate II in a potassium permanganate solution with the mass fraction of 0.1-0.15% for 20-24 hours, drying, adding shell powder, calcining at 600-800 ℃ in an oxygen-free environment, soaking in a phosphoric acid solution with the mass fraction of 6-8% for 6-8 hours, and drying to obtain the peanut meal biochar.
2. The fermentation medium of claim 1, wherein in the step 1, the inoculation amount of the aspergillus oryzae spore suspension is 0.2-0.25% of the volume of homogenate I, the inoculation amount of the pichia pastoris seed liquid is 0.1-0.3% of the volume of homogenate I, the number of spores in the aspergillus oryzae spore suspension is 1X 10 4~1×105/mL, and the OD value of the pichia pastoris seed liquid is 0.5-0.7.
3. The fermentation medium of claim 1, wherein in the step 1, the addition amount of stevioside is 5-8% of the mass of peanut meal, the addition amount of lipase is 0.3-0.6% of the mass of peanut meal, and the enzyme activity of the lipase is 20000U/g.
4. The fermentation medium of claim 1, wherein in step 2, the hemicellulase is added in an amount of 0.1 to 0.15% of the mass of the precipitate I, and the enzyme activity of the hemicellulase is 20000U/g.
5. The fermentation medium of claim 1, wherein in step 2, the amount of subtilisin added is 0.08-0.1% of the mass of precipitate I, 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 of claim 1, wherein in step 2, the volume ratio of supernatant II to sodium chloride solution is 1 (0.6-0.8).
7. The fermentation medium of claim 1, wherein in step 3, the shell powder is added in an amount of 0.2 to 0.25% by mass of precipitate II.
8. The fermentation medium of claim 1, wherein the method of preparing gelatinized potato starch comprises the steps of:
adding water into potato starch to obtain suspension, heating at 60-70deg.C to transparent, cooling to room temperature, drying, and pulverizing to obtain gelatinized potato starch.
9. A method for producing avermectin using the fermentation medium of any one of claims 1-8, comprising the steps of:
step A, preparing a basic culture solution and a fluid replacement, namely uniformly mixing peanut meal extract I, peanut meal biochar, gelatinized potato starch, yeast extract powder, magnesium sulfate heptahydrate, diammonium phosphate and calcium lactate with water, and performing ultrasonic treatment for 30-40min to prepare the basic culture solution;
and B, inoculating a production strain into the basic culture solution obtained in the step A, standing and culturing for 20-24 hours, culturing for 48-72 hours under a stirring state, adding a fluid replacement, and culturing for 144-168 hours under the stirring state to obtain the avermectin-containing fermentation liquor.
10. The method of claim 9, wherein the volume ratio of basal medium to replacement medium in step B is 1 (0.3-0.5).
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