CN109913503B - Method for producing tetraene compound by utilizing streptomyces albidoflauvs Ah11601 - Google Patents
Method for producing tetraene compound by utilizing streptomyces albidoflauvs Ah11601 Download PDFInfo
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Abstract
A method for producing tetraene compound by utilizing Streptomyces albidoflauvs Ah11601 comprises the following steps: taking 10-70g of soluble starch, putting the soluble starch into 900mL of water for gelatinization, then adding 10-50g of glucose, 2-6g of yeast extract, 2-6g of peptone, 2-6g of monopotassium phosphate, 1-4g of magnesium sulfate, 0.5-2g of sodium chloride and 0.5-2g of calcium carbonate, stirring for dissolution, and then adding water to fix the volume to 1000mL; adjusting pH and sterilizing to obtain a fermentation culture medium for later use; inoculating the activated Streptomyces albogriseus Ah11601 single colony into 100mL of activated seed culture medium, performing shake fermentation culture at 26 ℃ and 140rpm for 48h, then inoculating into a fermentation culture medium, wherein the inoculation amount accounts for 5% of the volume of the culture medium, and then performing shake culture at 27 ℃ and 180rpm for 144 h. The method has simple and easy operation steps and high yield of the tetraene compound.
Description
Technical Field
The invention relates to a method for producing a tetraene compound by utilizing streptomyces albidoflauvs Ah 11601.
Background
Actinomycetes are a type of gram-positive bacteria with complex morphological differentiation, are widely distributed in various environments, and the generated tetraene compound can be used as an agricultural antibiotic, and has the advantages of high efficiency, low residue, environmental friendliness and the like. Especially streptomyces can generate a plurality of tetraene compounds, and has wide prospect for preventing and controlling plant diseases.
However, the fermentation production of various active substances by utilizing streptomyces is influenced by nutritional factors such as carbon source, nitrogen source, carbon-nitrogen ratio, mineral salt, trace elements, vitamins and the like on one hand and fermentation process conditions such as culture time, pH, temperature, lysogen and the like on the other hand, most of the existing methods for producing tetraene compounds by utilizing streptomyces are low in yield and complex in steps, and are not beneficial to large-scale production.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for producing tetraene compound by utilizing streptomyces albidoflauvs Ah11601, which has simple steps and high yield.
In order to solve the technical problem, the invention provides a method for producing tetraene compound by utilizing streptomyces albidoflauvs Ah11601, which comprises the following steps:
(1) Taking 10-70g of soluble starch, and heating and pasting in 900mL of deionized water to form uniform semitransparent starch pasting liquid;
(2) Adding 10-50g of glucose, 2-6g of yeast extract, 2-6g of peptone, 2-6g of potassium dihydrogen phosphate, 1-4g of magnesium sulfate, 0.5-2g of sodium chloride and 0.5-2g of calcium carbonate into the starch pasting solution prepared in the step (1), keeping the pasting temperature in the step (1), stirring and dissolving, then cooling to room temperature, and fixing the volume to 1000mL by using deionized water;
(3) Adding 0.1mol/L phosphoric acid solution into the liquid with the constant volume in the step (2), adjusting the pH value to 5.6-7, and then sterilizing at 121 ℃ for 20min to obtain a fermentation medium of streptomyces albidoflauvr Ah11601 for later use;
(4) Inoculating a single colony of activated streptomyces albidoflauvs Ah11601 into 100mL of activated seed culture medium, and performing shake fermentation culture at 26 ℃ and 140rpm for 48 hours to obtain a fresh seed solution;
(5) Inoculating the fresh seed liquid into the fermentation culture medium of the streptomyces albidoflauvs Ah11601 prepared in the step (3), wherein the inoculation amount accounts for 5% of the volume of the culture medium, and then performing shake cultivation for 144h at 27 ℃ and 180 rpm.
Preferably, 20-60g of soluble starch is taken in the step (1), 20-40g of glucose, 2-5g of yeast extract, 3-5g of peptone, 3-6g of monopotassium phosphate, 1-3g of magnesium sulfate, 0.5-1.5g of sodium chloride and 0.5-1.5g of calcium carbonate are taken in the step (2), and the pH value is adjusted to be 5.6-6.2 in the step (3).
Preferably, 40g of soluble starch is taken in the step (1), 30g of glucose, 4g of yeast extract, 4g of peptone, 4g of monopotassium phosphate, 2g of magnesium sulfate, 1g of sodium chloride and 1g of calcium carbonate are taken in the step (2), and the pH is adjusted to be 5.6 in the step (3).
Preferably, the activated seed culture medium comprises the following components in percentage by mass: 15% of glucose, 5% of starch, 0.5% of peptone, 0.5% of yeast powder, 0.5% of potassium dihydrogen phosphate, 0.2% of magnesium sulfate, 0.1% of sodium chloride and the balance of water, wherein the pH of the activated seed culture medium is 6.8-7.0, and the activated seed culture medium needs to be sterilized at 121 ℃ for 20min before inoculation.
For the sake of simplicity, the method for producing tetraene compounds by using Streptomyces albus Ah11601 is also referred to as the method.
The Streptomyces albidoflauvs Ah11601 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation number is CGMCC No.11363, the preservation date is 2015 9-11 days, the preservation unit address is No. 3 of Xilu 1 of Beijing Kogyo sunward, china academy of sciences microbial research institute.
The method has the advantages that: the method has simple and easy operation steps, high yield of the tetraene compound and short culture period, and is suitable for batch production to better solve the problem of plant diseases.
Detailed Description
The first embodiment is as follows:
a method for producing tetraene compound by utilizing Streptomyces albidoflauvs Ah11601 comprises the following steps:
(1) Taking 10g of soluble starch, and placing the soluble starch in 900mL of deionized water for heating and pasting to form uniform semitransparent starch pasting liquid;
(2) Adding 10g of glucose, 2g of yeast extract, 2g of peptone, 2g of monopotassium phosphate, 1g of magnesium sulfate, 0.5g of sodium chloride and 0.5g of calcium carbonate into the starch pasting solution prepared in the step (1), keeping the pasting temperature in the step (1), stirring and dissolving, then cooling to room temperature, and fixing the volume to 1000mL by using deionized water;
(3) Adding 0.1mol/L phosphoric acid solution into the liquid with the constant volume in the step (2), adjusting the pH value to 6.8, and then sterilizing at 121 ℃ for 20min to obtain a fermentation medium of streptomyces albidoflauvs Ah11601 for later use;
(4) Inoculating a single colony of activated streptomyces albidoflauvs Ah11601 into 100mL of activated seed culture medium, and performing shake fermentation culture at 26 ℃ and 140rpm for 48 hours to obtain a fresh seed solution;
(5) Inoculating the fresh seed liquid into the fermentation culture medium of the streptomyces albidoflauvs Ah11601 prepared in the step (3), wherein the inoculation amount accounts for 5% of the volume of the culture medium, and then performing shake cultivation for 144h at 27 ℃ and 180 rpm.
Example two:
a method for producing tetraene compound by utilizing Streptomyces leucotrichia Ah11601 comprises the following steps:
(1) Taking 25g of soluble starch, and placing the soluble starch in 900mL of deionized water for heating and pasting to form uniform semitransparent starch pasting liquid;
(2) Adding 20g of glucose, 3g of yeast extract, 3g of peptone, 3g of potassium dihydrogen phosphate, 1.5g of magnesium sulfate, 0.75g of sodium chloride and 0.75g of calcium carbonate into the starch pasting liquid prepared in the step (1), keeping the pasting temperature in the step (1), stirring and dissolving, then cooling to room temperature, and fixing the volume to 1000mL by using deionized water;
(3) Adding 0.1mol/L phosphoric acid solution into the liquid with the constant volume in the step (2), adjusting the pH value to 6, and then sterilizing at 121 ℃ for 20min to obtain a fermentation medium of streptomyces albidoflauvs Ah11601 for later use;
(4) Inoculating a single colony of activated streptomyces albidoflauvs Ah11601 into 100mL of activated seed culture medium, and performing shake fermentation culture at 26 ℃ and 140rpm for 48 hours to obtain a fresh seed solution;
(5) Inoculating the fresh seed liquid into the fermentation culture medium of the streptomyces albidoflauvs Ah11601 prepared in the step (3), wherein the inoculation amount accounts for 5% of the volume of the culture medium, and then performing shake cultivation for 144h at 27 ℃ and 180 rpm.
Example three:
a method for producing tetraene compound by utilizing Streptomyces albidoflauvs Ah11601 comprises the following steps:
(1) Heating 25g of soluble starch in 900mL of deionized water for gelatinization to form uniform semitransparent starch gelatinization liquid;
(2) Adding 20g of glucose, 4g of yeast extract, 4g of peptone, 4g of monopotassium phosphate, 2g of magnesium sulfate, 1g of sodium chloride and 1g of calcium carbonate into the starch pasting liquid prepared in the step (1), keeping the pasting temperature in the step (1), stirring and dissolving, then cooling to room temperature, and fixing the volume to 1000mL by using deionized water;
(3) Adding 0.1mol/L phosphoric acid solution into the liquid with the constant volume in the step (2), adjusting the pH value to 5.6, and then sterilizing at 121 ℃ for 20min to obtain a fermentation medium of streptomyces albidoflauvs Ah11601 for later use;
(4) Inoculating a single colony of activated streptomyces albidoflauvs Ah11601 into 100mL of activated seed culture medium, and performing shake fermentation culture at 26 ℃ and 140rpm for 48 hours to obtain a fresh seed solution;
(5) Inoculating the fresh seed liquid into the fermentation culture medium of the streptomyces albidoflauvs Ah11601 prepared in the step (3), wherein the inoculation amount accounts for 5% of the volume of the culture medium, and then performing shake cultivation for 144h at 27 ℃ and 180 rpm.
Example four:
a method for producing tetraene compound by utilizing Streptomyces leucotrichia Ah11601 comprises the following steps:
(1) Taking 70g of soluble starch, and placing the soluble starch in 900mL of deionized water for heating and pasting to form uniform semitransparent starch pasting liquid;
(2) Adding 50g of glucose, 6g of yeast extract, 6g of peptone, 6g of monopotassium phosphate, 4g of magnesium sulfate, 2g of sodium chloride and 2g of calcium carbonate into the starch pasting liquid prepared in the step (1), keeping the pasting temperature in the step (1), stirring and dissolving, then cooling to room temperature, and fixing the volume to 1000mL by using deionized water;
(3) Adding 0.1mol/L phosphoric acid solution into the liquid with constant volume in the step (2), adjusting the pH to 7, and then sterilizing at 121 ℃ for 20min to obtain a fermentation medium of streptomyces albidoflavus Ah11601 for later use;
(4) Inoculating a single colony of activated streptomyces albidoflauvs Ah11601 into 100mL of activated seed culture medium, and performing shake fermentation culture at 26 ℃ and 140rpm for 48 hours to obtain a fresh seed solution;
(5) Inoculating the fresh seed liquid into the fermentation culture medium of the streptomyces albidoflauvs Ah11601 prepared in the step (3), wherein the inoculation amount accounts for 5% of the volume of the culture medium, and then performing shake cultivation for 144h at 27 ℃ and 180 rpm.
In order to verify the actual effect of the method, the content of the tetraene compound is determined by aiming at the fermentation liquid obtained by the culture in the first embodiment, the second embodiment and the third embodiment:
taking 1mL of the fermentation liquid prepared finally in the three examples, centrifuging at 10000rpm for 20min, taking the supernatant, using the supernatant for HPLC to determine the content of the tetraene compound (the detector used in the HPLC detection is an ultraviolet absorption detector), and further calculating the content of the tetraene compound in the fermentation liquid obtained in each example, wherein the test results are as follows:
EXAMPLE one the fermentation broth obtained was found to have a tetraene compound content of 4.8mg/L.
The content of the tetraene compound measured in the fermentation liquid obtained in example two was 6.7mg/L.
The fermentation liquid obtained in example three had a tetraene compound content of 8.2mg/L.
Therefore, the method has the advantages of high yield of the tetraene compound, simple operation, short culture period and suitability for batch production.
Claims (4)
1. A method for producing tetraene compounds by using Streptomyces albogriseus Ah11601 (Streptomyces albogriseus) is characterized by comprising the following steps of:
(1) Taking 10-70g of soluble starch, and heating and pasting in 900mL of deionized water to form uniform semitransparent starch pasting liquid;
(2) Adding 10-50g of glucose, 2-6g of yeast extract, 2-6g of peptone, 2-6g of potassium dihydrogen phosphate, 1-4g of magnesium sulfate, 0.5-2g of sodium chloride and 0.5-2g of calcium carbonate into the starch pasting solution prepared in the step (1), keeping the pasting temperature in the step (1), stirring and dissolving, then cooling to room temperature, and fixing the volume to 1000mL by using deionized water;
(3) Adding 0.1mol/L phosphoric acid solution into the liquid with the constant volume in the step (2), adjusting the pH value to 5.6-7, and then sterilizing at 121 ℃ for 20min to obtain a fermentation medium of streptomyces albidoflauvr Ah11601 for later use;
(4) Inoculating a single colony of activated streptomyces albidoflauvs Ah11601 into 100mL of activated seed culture medium, and performing shake fermentation culture at 26 ℃ and 140rpm for 48 hours to obtain a fresh seed solution;
(5) Inoculating a fresh seed solution into the fermentation culture medium of the streptomyces albidoflauvs Ah11601 prepared in the step (3), wherein the inoculation amount accounts for 5% of the volume of the culture medium, and then performing shake cultivation for 144h at 27 ℃ and 180 rpm;
the preservation number of the streptomyces albidoflauvs Ah11601 in the step (4) is CGMCC No.11363.
2. The method for producing tetraene compound by utilizing Streptomyces albuginea Ah11601 as claimed in claim 1, wherein: 20-60g of soluble starch is taken in the step (1), 20-40g of glucose, 2-5g of yeast extract, 3-5g of peptone, 3-6g of potassium dihydrogen phosphate, 1-3g of magnesium sulfate, 0.5-1.5g of sodium chloride and 0.5-1.5g of calcium carbonate are taken in the step (2), and the pH value is adjusted to be 5.6-6.2 in the step (3).
3. The method for producing tetraene compounds by utilizing Streptomyces albogriseus Ah11601, according to claim 1, wherein: 40g of soluble starch is taken in the step (1), 30g of glucose, 4g of yeast extract, 4g of peptone, 4g of monopotassium phosphate, 2g of magnesium sulfate, 1g of sodium chloride and 1g of calcium carbonate are taken in the step (2), and the pH is adjusted to be 5.6 in the step (3).
4. The method for producing tetraene compound by using Streptomyces albuginea Ah11601 according to any one of claims 1 to 3, wherein: the activated seed culture medium comprises the following components in percentage by mass: 15% of glucose, 5% of starch, 0.5% of peptone, 0.5% of yeast powder, 0.5% of potassium dihydrogen phosphate, 0.2% of magnesium sulfate, 0.1% of sodium chloride and the balance of water, wherein the pH of the activated seed culture medium is 6.8-7.0, and the activated seed culture medium is sterilized at 121 ℃ for 20min before inoculation.
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