CN108774637B - Method for producing nemadectin - Google Patents
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Abstract
The invention provides a method for efficiently producing nemadectin, which adopts L-valine-resistant streptomyces glaucescens to perform fermentation production in the presence of L-valine, solves the problem that the fermentation yield is slowly increased in the middle and later stages of fermentation in the traditional process, prolongs the fermentation period, and obviously improves the fermentation yield of the nemadectin.
Description
Technical Field
The invention relates to a microbial fermentation method, in particular to a method capable of improving the fermentation yield of nimustine.
Background
Nimustine (Nemadectin) is a sixteen-membered macrolide antibiotic, belongs to one member of the milbemycins, is produced by fermentation of Streptomyces cyaneogriseus sp. The structural formula of nimustine is:
moxidectin is used as an animal anthelmintic since the middle of the 80 th century, can efficiently kill nematodes, ectoparasites and the like, has good safety to animals, is a novel broad-spectrum, efficient and safe macrolide anthelmintic antibiotic, is widely used in veterinary clinic at present, and has increasingly large market demand. Therefore, a method capable of producing nimustine with high efficiency is desired.
Disclosure of Invention
In order to improve the production efficiency of nemadectin, the invention provides a method for producing nemadectin with high efficiency.
The method for producing the nimustine comprises the following steps: l-valine is added to a fermentation broth containing Streptomyces cyanogriseus sp. Wherein the streptomyces glaucescens is L-valine-resistant streptomyces glaucescens; during the fermentation process, the mass concentration of the growth factor of the streptomyces glaucescens in the fermentation liquor is controlled to be maintained at 0.1-8%.
In a preferred embodiment, the Streptomyces cyaneus (Streptomyces cyaneogyreius sp. Noncyanogenus) KS12-08 is deposited at the general microbiological center of China Committee for culture Collection of microorganisms at 2017, 2 and 15 days, with the deposit number of CGMCC No. 13661.
In a preferred embodiment, the L-valine is added in a preferred amount of 0.01% to 0.1%, more preferably 0.02% to 0.08%, by weight of the fermentation broth, such as: 0.04%, 0.05%, 0.06%, 0.07%.
In a preferred embodiment, during the fermentation process of the streptomyces glaucomatosus, the L-valine is added every day, and the weight ratio of the L-valine added every day to the fermentation broth is preferably 0.01% -0.1%, more preferably 0.02% -0.08%, such as: 0.04%, 0.05%, 0.06%, 0.07%.
In a preferred embodiment, L-valine is added after the completion of the primary metabolism of Streptomyces glaucomatosus in the fermentation broth.
In a preferred embodiment, the mass concentration of the growth factor of Streptomyces glaucescens in the fermentation broth is controlled to be maintained at 0.2% to 6%, more preferably 0.4% to 4%, more preferably 0.5% to 2%, more preferably 0.7% to 1.5%, such as 1%, 1.2%, at least during the fermentation after addition of L-valine.
In a preferred embodiment, the fermentation process is supplemented with the growth factor of Streptomyces glaucomatosus when the mass concentration of the growth factor of Streptomyces glaucomatosus is less than 1.5%.
The streptomyces glaucescens growth factor is preferably one or more of monosaccharide, disaccharide or oligosaccharide. Wherein, the monosaccharide can be preferably one or more of threose, xylose, glucose, fructose, mannose and galactose; the disaccharide can be one or more of sucrose, maltose, isomaltose, lactose and trehalose; the oligosaccharide is preferably one or more of fructo-oligosaccharide, xylo-oligosaccharide, maltose-oligosaccharide and isomaltose-oligosaccharide.
In a preferred embodiment, the fermentation temperature during the fermentation is preferably 20-35 deg.C, more preferably 25-33 deg.C, such as 28 deg.C, 30 deg.C.
In a preferred embodiment, the Streptomyces glaucescens seed liquid is inoculated into a fermentation medium and cultured to obtain the fermentation liquid.
In a preferred embodiment, the fermentation medium comprises, based on the total weight of the fermentation medium: 8% of glucose, 4% of maltodextrin, 2.8% of soybean cake powder, 0.5% of yeast powder, 0.1% of magnesium sulfate, 0.01% of zinc sulfate and 0.4% of calcium carbonate.
More preferably, the glucose is supplemented when the glucose mass concentration in the fermentation broth is below 1.5%.
In a preferred embodiment, the Streptomyces glaucescens spores are inoculated into a seed culture medium to obtain a seed solution.
In a preferred embodiment, the seed medium comprises, based on the total weight of the seed medium: 2.0 percent of maltodextrin, 1.0 percent of glucose, 1.0 percent of yeast powder, 1.5 percent of soybean cake powder, 0.1 percent of magnesium sulfate, 0.1 percent of dipotassium phosphate and 0.2 percent of calcium carbonate.
In a preferred embodiment, the Streptomyces glaucomatosus is prepared into a spore suspension, cultured in a medium containing L-valine, further subjected to resistance selection to obtain a strain tolerant to L-valine, and cultured to obtain spores.
The method for producing the nimustine solves the problem that the fermentation yield in the middle and later stages of the fermentation is slowly increased in the traditional process, prolongs the fermentation period and obviously improves the fermentation yield of the nimustine.
Detailed Description
Culturing the initial strain with slant culture, preparing spore suspension with slant strain, and diluting until spore concentration is 108Adding diethyl sulfate solution into the suspension, placing the suspension in a shaking table for shaking culture at 28 ℃, taking out 5ml of spore suspension by using a pipette, placing the spore suspension in a culture dish with the diameter of 6cm, and placing a stirrer. Then the mutagenesis is carried out by irradiating an ultraviolet lamp tube.
Diluting the spore suspension subjected to ultraviolet mutagenesis into a certain concentration gradient under a red lamp, respectively taking 0.2ml of the spore suspension, coating the spore suspension on a culture medium plate, culturing the culture medium plate at 28 ℃ in the dark for 7 to 8 days, selecting a single colony, and screening to obtain a strain which is resistant to L-valine and is named as Streptomyces glaucomatosus (Streptomyces cyanogriseus sp. Noncyanogenus) KS12-08, wherein the strain is preserved in the general microbiological center of China Committee for culture Collection of microorganisms, the preservation date is 2017, 2 and 15 days, and the preservation number is CGMCC No. 13661.
The detection result of the 16S rDNA sequence shows that the mutant strain CGMCC No.13661 belongs to streptomyces coelicolor. The mutant strain CGMCC No.13661 has short spore filament, compact 2-3 circles of spiral shape, and smooth surface, and the spore is in the shape of oval to short column; in agar medium of gram formula synthesis No. 1: the gas silk is powder, and the basic silk is changed into grey blue from initial colorless; is resistant to L-valine.
Example 1
Step 1, inoculating streptomyces glaucescens KS12-08 to a slant culture medium, and culturing for 7-8 days at the temperature of 28-30 ℃ and the humidity of 20-60% to obtain mature slant strains; the slant culture medium comprises: 1.0% of starch, 0.5% of glucose, 0.4% of yeast powder, 0.05% of magnesium sulfate, 0.05% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and 2.0% of agar;
step 2, scraping a proper amount of spores from mature slant strains, inoculating the spores into a liquid seed culture medium, and culturing for 26-30 hours at the temperature of 28-30 ℃ and the stirring rotation speed of 200-500 rpm to obtain mature seed liquid; the seed culture medium comprises: 2.0% of maltodextrin, 1.0% of glucose, 1.0% of yeast powder, 1.5% of soybean cake powder, 0.1% of magnesium sulfate, 0.1% of dipotassium hydrogen phosphate and 0.2% of calcium carbonate;
step 3, inoculating the mature seed liquid into a fermentation culture medium according to the inoculation amount of 8-12%, and performing fermentation culture under the conditions that the temperature is 28-30 ℃, the stirring speed is 200-600 rpm, the tank pressure is 0.02-0.06 MPa, and the dissolved oxygen in the tank is more than or equal to 30%; the fermentation medium comprises: 8% of glucose, 4% of maltodextrin, 2.8% of soybean cake powder, 0.5% of yeast powder, 0.1% of magnesium sulfate, 0.01% of zinc sulfate and 0.4% of calcium carbonate;
preparing the L-valine into a solution with a proper concentration, sterilizing, and supplementing the L-valine accounting for 0.05 wt% of the fermentation liquor into the fermentation liquor every day in the fermentation process.
Example 2
Step 1, inoculating streptomyces glaucescens KS12-08 to a slant culture medium, and culturing for 7-8 days at the temperature of 28-30 ℃ and the humidity of 20-60% to obtain mature slant strains; the slant culture medium comprises: 1.0% of starch, 0.5% of glucose, 0.4% of yeast powder, 0.05% of magnesium sulfate, 0.05% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and 2.0% of agar;
step 2, scraping a proper amount of spores from mature slant strains, inoculating the spores into a liquid seed culture medium, and culturing for 26-30 hours at the temperature of 28-30 ℃ and the stirring rotation speed of 200-500 rpm to obtain mature seed liquid; the seed culture medium comprises: 2.0% of maltodextrin, 1.0% of glucose, 1.0% of yeast powder, 1.5% of soybean cake powder, 0.1% of magnesium sulfate, 0.1% of dipotassium hydrogen phosphate and 0.2% of calcium carbonate;
step 3, inoculating the mature seed liquid into a fermentation culture medium according to the inoculation amount of 8-12%, and performing fermentation culture under the conditions that the temperature is 28-30 ℃, the stirring speed is 200-600 rpm, the tank pressure is 0.02-0.06 MPa, and the dissolved oxygen in the tank is more than or equal to 30%; the fermentation medium comprises: 8% of glucose, 4% of maltodextrin, 2.8% of soybean cake powder, 0.5% of yeast powder, 0.1% of magnesium sulfate, 0.01% of zinc sulfate and 0.4% of calcium carbonate;
when the fermentation time is 48 hours, 0.05 wt% of L-valine is supplemented into the fermentation liquor every day, the L-valine is prepared into a solution with the concentration of 5 wt%, and after sterilization, the L-valine solution is supplemented into a fermentation tank according to 1 wt% of the volume of the fermentation liquor every day.
Example 3
Step 1, inoculating streptomyces glaucescens KS12-08 to a slant culture medium, and culturing for 7-8 days at the temperature of 28-30 ℃ and the humidity of 20-60% to obtain mature slant strains; the slant culture medium comprises: 1.0% of starch, 0.5% of glucose, 0.4% of yeast powder, 0.05% of magnesium sulfate, 0.05% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and 2.0% of agar;
step 2, scraping a proper amount of spores from mature slant strains, inoculating the spores into a liquid seed culture medium, and culturing for 26-30 hours at the temperature of 28-30 ℃ and the stirring rotation speed of 200-500 rpm to obtain mature seed liquid; the seed culture medium comprises: 2.0% of maltodextrin, 1.0% of glucose, 1.0% of yeast powder, 1.5% of soybean cake powder, 0.1% of magnesium sulfate, 0.1% of dipotassium hydrogen phosphate and 0.2% of calcium carbonate;
step 3, inoculating the mature seed liquid into a fermentation culture medium according to the inoculation amount of 8-12%, and performing fermentation culture under the conditions that the temperature is 28-30 ℃, the stirring speed is 200-600 rpm, the tank pressure is 0.02-0.06 MPa, and the dissolved oxygen in the tank is more than or equal to 30%; the fermentation medium comprises: 8% of glucose, 4% of maltodextrin, 2.8% of soybean cake powder, 0.5% of yeast powder, 0.1% of magnesium sulfate, 0.01% of zinc sulfate and 0.4% of calcium carbonate;
when the fermentation time is 48 hours, 0.05 wt% of L-valine is supplemented into the fermentation liquor every day, the L-valine is prepared into a solution with the concentration of 5 wt%, and after sterilization, the L-valine solution is supplemented into a fermentation tank according to 1 wt% of the volume of the fermentation liquor every day.
Sampling and measuring the concentration of glucose in the fermentation liquid on the tank, starting to supplement the glucose solution when the concentration of the glucose is reduced to be below 1.5%, maintaining the concentration of the glucose in the fermentation liquid on the tank to be between 0.5 and 2.0%, and stopping supplementing the glucose 12 hours before the tank is placed.
And finishing fermentation after fermentation is carried out for 240-260 hours to obtain the fermentation liquor containing the nimustine.
Example 4
Step 1, inoculating streptomyces glaucescens KS12-08 to a slant culture medium, and culturing for 7-8 days at the temperature of 28-30 ℃ and the humidity of 20-60% to obtain mature slant strains; the slant culture medium comprises: 1.0% of starch, 0.5% of glucose, 0.4% of yeast powder, 0.05% of magnesium sulfate, 0.05% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and 2.0% of agar;
and 2, scraping spores on a grown strain inclined plane, wherein the spores are on the inclined plane of about 0.5cm multiplied by 0.5cm, inoculating the spores into a 500ml seed shaking bottle, wherein the seed shaking bottle is filled with 50ml, and culturing the inoculated seed shaking bottle on a shaking table with the rotating speed of 200rpm for 28 hours at the temperature of 28 ℃.
And 3, inoculating the cultured shake flask seeds to a 5L glass fermentation tank according to the inoculation amount of 8%, wherein the loading amount of the fermentation tank is 2.5L, the culture condition of the fermentation tank is that the tank temperature is 28 ℃, the ventilation volume is 0.8vvm, the stirring speed is 200rpm, and the Dissolved Oxygen (DO) value on the fermentation tank is controlled to be more than or equal to 30% by increasing the stirring speed in the fermentation process.
When the fermentation culture is carried out for 48 hours, the sterilized L-valine solution with the concentration of 5 wt% is supplemented once a day, 25ml is supplemented each time, namely the L-valine is supplemented once each time when the fermentation culture is carried out for 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours and 216 hours.
When the fermentation culture is carried out for 150 hours, feeding glucose supplementation solution, controlling the glucose concentration in the fermentation liquor to be between 0.5 and 2.0 percent by adjusting the glucose supplementation speed, and stopping glucose supplementation at the 228 th hour.
And finishing the culture after the fermentation culture is finished for 240 hours to obtain the fermentation liquor containing the nimustine.
Example 5
Step 1, inoculating streptomyces glaucescens KS12-08 to a slant culture medium, and culturing for 7-8 days at the temperature of 28-30 ℃ and the humidity of 20-60% to obtain mature slant strains; the slant culture medium comprises: 1.0% of starch, 0.5% of glucose, 0.4% of yeast powder, 0.05% of magnesium sulfate, 0.05% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and 2.0% of agar;
and 2, scraping spores on a grown strain inclined plane, wherein the spores are on the inclined plane of about 0.5cm multiplied by 0.5cm, inoculating the spores into a 500ml seed shaking bottle, wherein the seed shaking bottle is filled with 50ml, and culturing the inoculated seed shaking bottle on a shaking table with the rotating speed of 200rpm for 28 hours at the temperature of 28 ℃.
And 3, inoculating the cultured shake flask seeds to a 10L stainless steel seed tank according to the inoculation amount of 1%, wherein the loading amount of the seed tank is 5.0L, the culture conditions of the seed tank are that the tank temperature is 28 ℃, the ventilation volume is 1.0vvm, the stirring speed is 200rpm, the Dissolved Oxygen (DO) value on the seed tank is controlled to be more than or equal to 30% by increasing the stirring speed in the culture process, and the culture time of the seed tank is 24 h.
And 4, inoculating the seed liquid of the cultured seeding tank to a 50L fermentation tank according to the inoculation amount of 10%, wherein the loading amount of the fermentation tank is 30L, the culture condition of the fermentation tank is that the tank temperature is 28 ℃, the ventilation volume is 0.8vvm, the stirring rotation speed is 200rpm, and the Dissolved Oxygen (DO) value on the fermentation tank is controlled to be more than or equal to 30% by increasing the stirring rotation speed in the culture process.
When the fermentation culture is carried out for 48 hours, the sterilized L-valine solution with the concentration of 5 wt% is supplemented, the supplement amount is 300 ml/day in a constant-speed flow supplement manner, and the supplement is stopped when the time is 228 hours.
When the fermentation culture is carried out for 150 hours, feeding glucose supplementation solution, controlling the glucose concentration in the fermentation liquor to be between 0.5 and 2.0 percent by adjusting the glucose supplementation speed, and stopping glucose supplementation at the 228 th hour.
And finishing the culture after the fermentation culture is finished for 240 hours to obtain the fermentation liquor containing the nimustine.
Comparative example 1
Step 1, inoculating initial streptomyces coelicolor (a strain which is not subjected to ultraviolet lamp light irradiation mutagenesis) to a slant culture medium, and culturing for 7-8 days at the temperature of 28-30 ℃ and the humidity of 20-60% to obtain mature slant strains; the slant culture medium comprises: 1.0% of starch, 0.5% of glucose, 0.4% of yeast powder, 0.05% of magnesium sulfate, 0.05% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and 2.0% of agar;
and 2, scraping spores on a grown strain inclined plane, wherein the spores are on the inclined plane of about 0.5cm multiplied by 0.5cm, inoculating the spores into a 500ml seed shaking bottle, wherein the seed shaking bottle is filled with 50ml, and culturing the inoculated seed shaking bottle on a shaking table with the rotating speed of 200rpm for 28 hours at the temperature of 28 ℃.
And 3, inoculating the cultured shake flask seeds to a 10L stainless steel seed tank according to the inoculation amount of 1%, wherein the loading amount of the seed tank is 5.0L, the culture conditions of the seed tank are that the tank temperature is 28 ℃, the ventilation volume is 1.0vvm, the stirring speed is 200rpm, the Dissolved Oxygen (DO) value on the seed tank is controlled to be more than or equal to 30% by increasing the stirring speed in the culture process, and the culture time of the seed tank is 24 h.
And 4, inoculating the seed liquid of the cultured seeding tank to a 50L fermentation tank according to the inoculation amount of 10%, wherein the loading amount of the fermentation tank is 30L, the culture condition of the fermentation tank is that the tank temperature is 28 ℃, the ventilation volume is 0.8vvm, the stirring rotation speed is 200rpm, and the Dissolved Oxygen (DO) value on the fermentation tank is controlled to be more than or equal to 30% by increasing the stirring rotation speed in the culture process.
When the fermentation culture is carried out for 48 hours, the sterilized L-valine solution with the concentration of 5 wt% is supplemented, the supplement amount is 300 ml/day in a constant-speed flow supplement manner, and the supplement is stopped when the time is 228 hours.
And finishing the culture after the fermentation culture is finished for 240 hours to obtain the fermentation liquor containing the nimustine.
Comparative example 2
Step 1, inoculating initial streptomyces coelicolor (a strain which is not subjected to ultraviolet lamp light irradiation mutagenesis) to a slant culture medium, and culturing for 7-8 days at the temperature of 28-30 ℃ and the humidity of 20-60% to obtain mature slant strains; the slant culture medium comprises: 1.0% of starch, 0.5% of glucose, 0.4% of yeast powder, 0.05% of magnesium sulfate, 0.05% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and 2.0% of agar;
and 2, scraping spores on a grown strain inclined plane, wherein the spores are on the inclined plane of about 0.5cm multiplied by 0.5cm, inoculating the spores into a 500ml seed shaking bottle, wherein the seed shaking bottle is filled with 50ml, and culturing the inoculated seed shaking bottle on a shaking table with the rotating speed of 200rpm for 28 hours at the temperature of 28 ℃.
And 3, inoculating the cultured shake flask seeds to a 10L stainless steel seed tank according to the inoculation amount of 1%, wherein the loading amount of the seed tank is 5.0L, the culture conditions of the seed tank are that the tank temperature is 28 ℃, the ventilation volume is 1.0vvm, the stirring speed is 200rpm, the Dissolved Oxygen (DO) value on the seed tank is controlled to be more than or equal to 30% by increasing the stirring speed in the culture process, and the culture time of the seed tank is 24 h.
And 4, inoculating the seed liquid of the cultured seeding tank to a 50L fermentation tank according to the inoculation amount of 10%, wherein the loading amount of the fermentation tank is 30L, the culture condition of the fermentation tank is that the tank temperature is 28 ℃, the ventilation volume is 0.8vvm, the stirring rotation speed is 200rpm, and the Dissolved Oxygen (DO) value on the fermentation tank is controlled to be more than or equal to 30% by increasing the stirring rotation speed in the culture process.
When the fermentation culture is carried out for 48 hours, the sterilized L-valine solution with the concentration of 5 wt% is supplemented, the supplement amount is 300 ml/day in a constant-speed flow supplement manner, and the supplement is stopped when the time is 228 hours.
When the fermentation culture is carried out for 150 hours, feeding glucose supplementation solution, controlling the glucose concentration in the fermentation liquor to be between 0.5 and 2.0 percent by adjusting the glucose supplementation speed, and stopping glucose supplementation at the 228 th hour.
And finishing the culture after the fermentation culture is finished for 240 hours to obtain the fermentation liquor containing the nimustine.
Comparative example 3
Step 1, inoculating initial streptomyces coelicolor (a strain which is not subjected to ultraviolet lamp light irradiation mutagenesis) to a slant culture medium, and culturing for 7-8 days at the temperature of 28-30 ℃ and the humidity of 20-60% to obtain mature slant strains; the slant culture medium comprises: 1.0% of starch, 0.5% of glucose, 0.4% of yeast powder, 0.05% of magnesium sulfate, 0.05% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and 2.0% of agar;
and 2, scraping spores on a grown strain inclined plane, wherein the spores are on the inclined plane of about 0.5cm multiplied by 0.5cm, inoculating the spores into a 500ml seed shaking bottle, wherein the seed shaking bottle is filled with 50ml, and culturing the inoculated seed shaking bottle on a shaking table with the rotating speed of 200rpm for 28 hours at the temperature of 28 ℃.
And 3, inoculating the cultured shake flask seeds to a 10L stainless steel seed tank according to the inoculation amount of 1%, wherein the loading amount of the seed tank is 5.0L, the culture conditions of the seed tank are that the tank temperature is 28 ℃, the ventilation volume is 1.0vvm, the stirring speed is 200rpm, the Dissolved Oxygen (DO) value on the seed tank is controlled to be more than or equal to 30% by increasing the stirring speed in the culture process, and the culture time of the seed tank is 24 h.
And 4, inoculating the seed liquid of the cultured seeding tank to a 50L fermentation tank according to the inoculation amount of 10%, wherein the loading amount of the fermentation tank is 30L, the culture condition of the fermentation tank is that the tank temperature is 28 ℃, the ventilation volume is 0.8vvm, the stirring rotation speed is 200rpm, and the Dissolved Oxygen (DO) value on the fermentation tank is controlled to be more than or equal to 30% by increasing the stirring rotation speed in the culture process.
And finishing the culture after the fermentation culture is finished for 240 hours to obtain the fermentation liquor containing the nimustine.
Table 1, comparison of the effects of the method for producing nimustine of the present invention and the comparative example
As can be seen from the comparison in Table 1, the strain used in the present application can significantly improve the fermentation yield of nimustine in the presence of L-valine, and the non-mutagenized strain (comparative examples 1-3) can cause the reduction of the fermentation yield of nimustine after the addition of L-valine. The reason is that the conventional streptomyces glaucomatosus has certain sensitivity to L-valine, and the L-valine can generate toxicity to the streptomyces glaucomatosus in the presence of the L-valine, so that the activity of the strain is reduced, and the fermentation efficiency is reduced
The applicant believes that the isopropyl group in the nimustine molecule is derived from L-valine, so that the addition of the L-valine in the fermentation broth can save the time and nutrition required by the bacteria to synthesize the L-valine, thereby improving the yield. For example, in examples 4-5 of the present invention, compared to comparative examples 1-3, the yield of nimustine produced by the method of the present invention was improved by 9-12% compared to the highest yield of the non-mutagenized strain.
Comparing example 1 with examples 2 and 3, it can be seen that the effect of adding L-valine to the initial fermentation medium is not significant, while after 48h of fermentation, the primary metabolism of the strain is substantially completed, and the secondary metabolism is predominant, at which time the effect of adding L-valine is better, especially the effect of adding small amounts in portions is better.
In comparison with examples 1 to 5, the present invention can further improve the fermentation efficiency by maintaining the concentration of growth factors such as glucose during the fermentation process.
It should be understood that the various media described in the above examples of the invention may be replaced by other media without technical difficulties for the person skilled in the art.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (9)
1. A method of producing nimustine comprising: adding L-valine into fermentation liquor containing streptomyces coelicolor for fermentation; the streptomyces glaucescens is L-valine-resistant streptomyces glaucescens; in the fermentation process, the mass concentration of the streptomyces glaucescens growth factor in the fermentation liquor is controlled to be maintained at 0.1-8%;
wherein the streptomyces glaucescens is preserved in the China general microbiological culture Collection center (CGMCC), the preservation date is 2017, 2 months and 15 days, and the preservation number is CGMCC No. 13661;
wherein the streptomyces glaucescens growth factor is one or more of monosaccharide, disaccharide or oligosaccharide.
2. The method for producing nemadectin according to claim 1, wherein the added L-valine accounts for 0.01-0.1% of the weight of the fermentation broth.
3. The method for producing nemadectin according to claim 2, wherein L-valine is added every day in the fermentation process of the streptomyces glaucosus, and the weight proportion of the L-valine added every day in the fermentation broth is 0.01-0.1%.
4. The method for producing nemadectin according to claim 1, wherein L-valine is added after the Streptomyces glaucomatosus completes primary metabolism in the fermentation broth.
5. The method for producing nemadectin according to claim 1, wherein the mass concentration of the growth factor of streptomyces glaucescens in the fermentation broth is controlled to be maintained at 0.5-2% during the fermentation process after the addition of L-valine.
6. The method for producing nemadectin according to claim 5, wherein the feeding of the growth factor of Streptomyces glaucomatosus is performed when the mass concentration of the growth factor of Streptomyces glaucomatosus is less than 1.5% during the fermentation process.
7. The method for producing nimustine according to claim 1, wherein the monosaccharide is one or more of sucinose, xylose, glucose, fructose, mannose, galactose; the disaccharide is one or more of sucrose, maltose, isomaltose, lactose and trehalose; the oligosaccharide is one or more of fructo-oligosaccharide, xylo-oligosaccharide, maltose-oligosaccharide and isomaltose-oligosaccharide.
8. The method for producing nemadectin according to claim 1, wherein the seed solution of streptomyces glaucomatosus is inoculated into a fermentation medium and cultured to obtain the fermentation broth; taking the total weight of the fermentation medium as a reference, the fermentation medium comprises: 8% of glucose, 4% of maltodextrin, 2.8% of soybean cake powder, 0.5% of yeast powder, 0.1% of magnesium sulfate, 0.01% of zinc sulfate and 0.4% of calcium carbonate.
9. The method for producing nemadectin according to claim 8, wherein the fermentation temperature is 20-35 ℃ during the fermentation process.
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| CN102336796A (en) * | 2010-07-27 | 2012-02-01 | 北大方正集团有限公司 | Preparation method of nemadectin |
| CN104745654A (en) * | 2013-12-27 | 2015-07-01 | 牡丹江佰佳信生物科技有限公司 | Method and strain for preparing Nemadectin |
-
2018
- 2018-02-01 CN CN201810101138.9A patent/CN108774637B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102336796A (en) * | 2010-07-27 | 2012-02-01 | 北大方正集团有限公司 | Preparation method of nemadectin |
| CN104745654A (en) * | 2013-12-27 | 2015-07-01 | 牡丹江佰佳信生物科技有限公司 | Method and strain for preparing Nemadectin |
Non-Patent Citations (2)
| Title |
|---|
| Complete genome sequence of Streptomyces cyaneogriseus ssp. noncyanogenus, the thermotolerant producer of commercial antibiotics nemadectin;Haiyan Wang等;《Journal of Biotechnology》;20150401;第204卷;1-2 * |
| 农用抗生素尼莫克汀高产菌株的推理选育;王则;《中国优秀硕士学位论文全文数据库 农业科技辑》;20100315(第3期);D046-70，论文摘要及第36-51页 * |
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| CN108774637A (en) | 2018-11-09 |
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