METHOD FOR PREPARING STALLIMYCIN

Abstract

Provided is a method for preparing stallimycin. The method comprises: fermenting streptomyces in a fermentation medium comprising a carbon source, a nitrogen source and 3-hydroxy-4-aminobutyric acid, and adding vegetable oil into the fermentation medium during the fermentation.

Claims

1. A method for preparing stallimycin comprising the step of fermenting streptomyces that can produce stallimycin in a fermentation medium comprising an available carbon source, an available nitrogen source and 3-hydroxy-4-aminobutyric acid, and the step of adding vegetable oil into the fermentation medium during the fermentation.

2. The method according to claim 1, wherein the streptomyces is selected from the group consisting of Streptomyces distallicus NRRL NO. 2886, Streptomyces distallicus D32 or Streptomyces distallicus DZ206.

3. The method according to claim 1, wherein the weight concentration of the 3-hydroxy-4-aminobutyric acid in the fermentation medium is 0.005%-0.05%.

4. The method according to claim 1, wherein during the fermentation means at the time when the fermentation has been conducted for 48-120 hours.

5. The method according to claim 1, wherein the step of adding vegetable oil comprising adding vegetable oil in more than one addition, the interval between the additions of vegetable oil is once every 24 hours.

6. The method according to claim 5, wherein the weight ratio of the amount of vegetable oil added for each addition to the fermentation medium is 0.3-1.0%; the weight ratio of the total amount of the added vegetable oil to the fermentation medium is 1-4%.

7. The method according to claim 1, wherein the vegetable oil is selected from the group consisting of rapeseed oil, sunflower oil, peanut oil, soybean oil, olive oil or the mixtures thereof.

8. The method according to claim 1, wherein the weight concentration of the available carbon source in the fermentation medium is 4-10%; the weight concentration of the available nitrogen source in the fermentation medium is 2-7%.

9. The method according to claim 1, wherein the available carbon source is selected from the group consisting of lactose, maltose, dextrin, cassava flour, corn starch, glucose, wheat starch, potato starch, glycerol, vegetable oil or the mixtures thereof; the available nitrogen source is selected from soybean meal, yellow soybean cake meal, yeast powder, corn steep liquor, dried corn steep liquor powder, fish meal, peptone, peanut cake powder, cottonseed cake meal, bran, gluten powder, ammonium sulfate, ammonium phosphate, ammonium chloride, ammonium nitrate or the mixtures thereof.

10. The method according to claim 1, wherein the temperature of fermentation is 20-40 C., the time of fermentation is 144-192 hours, the pH of the fermentation medium is 6.0-9.0.

11. The method according to claim 3, wherein the weight concentration of the 3-hydroxy-4-aminobutyric acid in the fermentation medium is 0.01%.

12. The method according to claim 6, wherein the weight ratio of the total amount of the added vegetable oil to the fermentation medium is 1-4%.

13. The method according to claim 6, wherein the weight ratio of the amount of vegetable oil added for each time to the fermentation medium is 0.5%.

14. The method according to claim 6, wherein the weight ratio of the total amount of the added vegetable oil to the fermentation medium is 2%.

15. The method according to claim 7, wherein the vegetable oil is soybean oil.

16. The method according to claim 8, wherein the weight concentration of the available carbon source in the fermentation medium is 5-8%; the weight concentration of the available nitrogen source in the fermentation medium is 3-6%.

17. The method according to claim 16, wherein the weight concentration of the available carbon source in the fermentation medium is 6%; the weight concentration of the available nitrogen source in the fermentation medium is 5%.

18. The method according to claim 9, wherein the available carbon source is selected from the group consisting of glucose, dextrin, soybean oil or the mixtures thereof; the available nitrogen source is selected from the group consisting of soybean meal, gluten powder or the mixture thereof.

Description

EMBODIMENTS

[0022] The present invention will be further illustrated by the following examples, to let the present invention be fully understood by those skilled in the art. The examples are provided for illustrating the present invention but not limiting the present invention. Simple modification of the present invention according to the essence of the present invention all belong to the scope protected by the present invention. Unless stated otherwise, the percentages in the present invention are all weight percentages or weight percentage concentrations.

[0023] Streptomyces distallicus NRRL NO. 2886 was disclosed in U.S. Pat. No. 3,190,801, it can be obtained from Agricultural Research Service Culture Collection in the United States.

[0024] Streptomyces distallicus D32 and Streptomyces distallicus DZ206 were disclosed in Selection of High Distamycin Producing Strain, Jiang Shichun etc. Acta Laser Biology Sinica, 2014, 23(2), 189-192, it can be obtained from Zhejiang Hisun Pharmaceutical Co., LTD.

Comparative Example 1: Preparation of Stallimycin

[0025] (1) Strain: Streptomyces distallicus NRRL NO. 2886

(2) Preparation and Culture of Slant Pore

[0026] Formula of the medium (%): glucose 4.5, yeast powder 1.5, sodium chloride 0.3, magnesium sulfate heptahydrate 0.05, calcium carbonate 0.2, ammonium sulfate 0.05, EDTA 0.02, the pH before sterilization was 6.2, it was prepared with nature water, the loading quantity was 50 mL in a 250 mL eggplant-shaped bottle. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10 to 0.13 MPa for 30 minutes, it was laid aside and became a slant after suitable cooling for later use.

[0027] The spore solution of the stallimycin producing strain, Streptomyces distallicus NRRL NO. 2886 was uniformly coated on the above blank slant, and it was cultivated in an incubator with constant temperature at 292 C. for 6-12 days. After the spores were mature, they were ash grey.

(3) Preparation and Culture of Shake Flask Seed Culture Medium

[0028] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water, the loading quantity was 50 mL in a 500 mL triangular flask. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10-0.13 MPa for 30 minutes. After cooling, the medium blocks comprising spores were dug out and inoculated into the seed culture medium, then at the temperature of 292 C., they were cultured on a shaker with a rotation speed of 220 r/min for 40 hours, the concentration of mycelia was 7%, and the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained and had obvious branches and segments.

(4) Preparation and Culture of Shake Flask Fermentation Medium

[0029] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500n L triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the amount of inoculation was 10%, the culture temperature was 292 C., it was cultured on a shaker with a rotation speed of 220 r/min for 168 hours, the concentration of the mycelia in the bottles was about 35%, the pH was about 7.5, the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation medium, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 730 ug/mL by high performance liquid chromatography (HPLC).

Comparative Example 2: Preparation of Stallimycin

[0030] (1) Strain: Streptomyces distallicus D32

(2) Preparation and Culture of Slant Pore

[0031] Formula of the medium (%): glucose 4.5, yeast powder 1.5, sodium chloride 0.3, magnesium sulfate heptahydrate 0.05, calcium carbonate 0.2, ammonium sulfate 0.05, EDTA 0.02, the pH before sterilization was 6.2, it was prepared with nature water, the loading quantity was 50 mL in a 250 mL eggplant-shaped bottle. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10 to 0.13 MPa for 30 minutes, it was laid aside and became a slant after suitable cooling for later use.

[0032] The spore solution of the stallimycin producing strain, Streptomyces distallicus D32 was uniformly coated on the above blank slant, and it was cultivated in an incubator with constant temperature at 292 C. for 6-12 days. After the spores were mature, they were ash grey.

(3) Preparation and Culture of Shake Flask Seed Culture Medium

[0033] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water, the loading quantity was 50 mL in a 500 mL triangular flask. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10-0.13 MPa for 30 minutes. After cooling, the medium blocks comprising spores were dug out and inoculated into the seed culture medium, then at the temperature of 292 C., they were cultured on a shaker with a rotation speed of 220 r/min for 40 hours, the concentration of mycelia was 8%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained and they had obvious branches and segments.

(4) Preparation and Culture of Shake Flask Fermentation Medium

[0034] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, soybean oil 0.1. pH-16.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the amount of inoculation was 10%, the culture temperature was 292 C., it was cultured on a shaker with the rotation speed of 220 r/min for 168 hours, the concentration of the mycelia in the bottles was about 35%, the pH was about 79, the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation medium, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 1030 ug/mL by high performance liquid chromatography (HPLC).

Comparative Example 3: Preparation of Stallimycin

[0035] (1) Strain: Streptomyces distallicus DZ206

(2) Preparation and Culture of Slant Pore

[0036] Formula of the medium (%): glucose 4.5, yeast powder 1.5, sodium chloride 0.3, magnesium sulfate heptahydrate 0.05, calcium carbonate 0.2, ammonium sulfate 0.05, EDTA 0.02, the pH before sterilization was 6.2, it was prepared with nature water, the loading quantity was 50 mL in a 250 mL eggplant-shaped bottle. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10 to 0.13 MPa for 30 minutes, it was laid aside and became a slant after suitable cooling for later use.

[0037] The spore solution of the stallimycin producing strain, Streptomyces distallicus DZ206 was uniformly coated on the above blank slant, and it was cultivated in an incubator with constant temperature at 292 C. for 6-12 days. After the spores were mature, they were ash grey.

(3) Preparation and Culture of Shake Flask Seed Culture Medium

[0038] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water, the loading quantity was 50 mL in a 500 mL triangular flask. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10-0.13 MPa for 30 minutes. After cooling, the medium blocks comprising spores were dug out and inoculated into the seed culture medium, then at the temperature of 292 C., they were cultured on a shaker with a rotation speed of 220 r/min for 40 hours, the concentration of mycelia was 10%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained and they had obvious branches and segments.

(4) Preparation and Culture of Shake Flask Fermentation Medium

[0039] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the amount of inoculation was 10%, the culture temperature was 292 C., it was cultured on a shaker with the rotation speed of 220 r/min for 168 hours, the concentration of the mycelia in the bottle was about 35%, the pH was about 7.9, the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation medium, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of Stallimycin was determined to be 2420 ug/mL by high performance liquid chromatography (HPLC).

Comparative Example 4: Preparation of Stallimycin

[0040] Experiment with Small Fermentation Tank

(1) The Strain and the Preparation and Culture of Slant Pore are Seen in Comparative Example 3

(2) Preparation of Shake Flask Mycelia

[0041] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water, the loading quantity was 200 mL in a 1000 mL triangular flask. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10-0.13 MPa for 30 minutes. After cooling, the medium blocks comprising spores were dug out and inoculated into the seed culture medium, then at the temperature of 292 C., they were cultured on a shaker with a rotation speed of 220 r/min for 40 hours, the concentration of mycelia was about 14%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained and they had branches and segments.

(3) Preparation and Culture of Seed Tank

[0042] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water. The loading quantity was 65 L in a 100 L seed tank, the medium was subjected to sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, after cooling, the mycelia liquid of the shake flask was inoculated, then it was cultured at the temperature of 292 C., stirring speed 220 r/min, ventilation ratio 1:1 v/v/m for 40 hours, the concentration of the mycelia was 12%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained, thick and strong, and they had obvious branches and segments.

(4) Preparation and Culture of Fermentation Tank

[0043] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, soybean oil 0.1, pH6.2, it was prepared with drinking water. The loading quantity was 700 L in a 1000 L fermentation tank, the medium was subjected to sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, after cooling, the seed liquid was inoculated with the inoculation amount of 10%, the culture temperature was 292 C., the stirring rotation speed was 220 r/min, the ventilation ratio was 1:0.8 v/v/nm, the fermentation cycle was 168 hours, the concentration of the mycelia was about 40%, the appearance was dark yellow, pH 8.2, after the pretreatment of the fermentation liquid, the titer of stallimycin was determined to be 2470 ug/mL by high performance liquid chromatography (HPLC).

Example 1: Preparation of Stallimycin

[0044] (1) Strain: Streptomyces distallicus DZ206

(2) Preparation and Culture of Slant Spore

[0045] Formula of the medium (%): glucose 4.5, yeast powder 1.5, sodium chloride 0.3, magnesium sulfate heptahydrate 0.05, calcium carbonate 0.2, ammonium sulfate 0.05, EDTA 0.02, the pH before sterilization was 6.2, it was prepared with nature water, the loading quantity was 50 mL in a 500 mL triangular flask. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10 to 0.13 MPa for 30 minutes, it was laid aside and became a slant after suitable cooling for later use.

[0046] The spore solution of the stallimycin producing strain, Streptomyces distallicus DZ206 was uniformly coated on the above blank slant, and it was cultivated in an incubator with constant temperature at 292 C. for 6-12 days. After the spores were mature, they were ash grey.

(3) Preparation and Culture of Shake Flask Seed Culture Medium

[0047] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water, the loading quantity was 50 mL in a 500 mL triangular flask. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10-0.13 MPa for 30 minutes. After cooling, the medium blocks comprising spores were dug out and inoculated into the seed culture medium, then at the temperature of 292 C., they were cultured on a shaker with a rotation speed of 220 r/min for 40 hours, the concentration of mycelia was 12%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained and had obvious branches and segments.

(4) Preparation and Culture of Shake Flask Fermentation Medium

[0048] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.005, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the amount of inoculation was 10%, the culture temperature was 292 C., after culturing on a shaker with the rotation speed of 220 r/min for 48 hours, 0.5% soybean oil was added every 24 hours (in total, the amount of soybean oil added during the whole fermentation process was about 2%), it was cultivated for 168 hours and put in a bottle. The concentration of the mycelia of the fermentation medium was about 35%, the pH was about 7.8, and the appearance was dark yellow 3 fold (volume) of 80% ethanol was added to the fermentation medium, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 5430 ug/mL by high performance liquid chromatography (HPLC).

Example 2: Preparation of Stallimycin

[0049] Experiment with Small Fermentation Tank

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation of Shake Flask Mycelia

[0050] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water, the loading quantity was 200 mL in a 1000 mL triangular flask. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10-0.13 MPa for 30 minutes. After cooling, the medium blocks comprising spores were dug out and inoculated into the seed culture medium, then at the temperature of 292 C., they were cultured on a shaker with a rotation speed of 220 r/min for 40 hours, the concentration of mycelia was about 12%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained and had branches and segments.

(3) Preparation and Culture of Seed Tank

[0051] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water. The loading quantity was 65 L in a 100 L seed tank, the medium was subjected to sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, after cooling, the mycelia liquid of the shake flask was inoculated, then it was cultivated at the temperature of 292 C., stirring rotation speed 220 r/min, ventilation ratio 1:1 v/v/m for 40 hours, the concentration of the mycelia was 14%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained, thick and strong, and had obvious branches and segments.

(4) Preparation and Culture of Fermentation Tank

[0052] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3.0, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, soybean oil 0.1, HABA 0.01, pH6.2, it was prepared with drinking water. The loading quantity was 700 L in a 1000 L fermentation tank, the medium was subjected to sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, after cooling, the seed liquid was inoculated with the inoculation amount of 10%, the culture temperature was 292 C., the stirring rotation speed was 220 r/min, the ventilation ratio was 1:0.8 v/v/m. Soybean oil was added after 48 hours of culture, 0.3% was added once a day, in total, the amount of soybean oil supplemented during the whole fermentation process was 1.2%, the fermentation cycle was 168 hours, the concentration of the mycelia was about 40%, the appearance was dark yellow, pH 8.2, after the pretreatment of the fermentation liquid, the titer of stallimycin was determined to be 5360 ug/mL by high performance liquid chromatography (HPLC).

Example 3: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation of Shake Flask Mycelia

[0053] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water, the loading quantity was 200 mL in a 1000 mL triangular flask. The medium was subjected to sterilization at the temperature of 121-125 C. and at the pressure of 0.10-0.13 MPa for 30 minutes. After cooling, the medium blocks comprising spores were dug out and inoculated into the seed culture medium, then at the temperature of 292 C., they were cultured on a shaker with a rotation speed of 220 r/min for 40 hours, the concentration of mycelia was about 12%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained and had branches and segments.

(3) Preparation and Culture of Seed Tank

[0054] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH1-6.0, it was prepared with drinking water. The loading quantity was 65 L in a 100 L seed tank, the medium was subjected to sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, after cooling, the mycelia liquid of the shake flask was inoculated, then it was cultivated at the temperature of 292 C., stirring rotation speed 220 r/min, ventilation ratio 1:1 v/v/m for 40 hours, the concentration of the mycelia was 13%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained, thick and strong, and had obvious branches and segments.

(4) Preparation and Culture of Fermentation Medium

[0055] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1, pH6.2, it was prepared with drinking water. The loading quantity was 700 L in a 1000 L fermentation tank, the medium was subjected to sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, after cooling, the seed liquid was inoculated with the inoculation amount of 10%, the culture temperature was 292 C., the stirring speed was 220 r/min, the ventilation ratio was 1:0.8 v/v/m, soybean oil was added after 48 hours of culture, 0.5% was added once a day, in total, the amount of soybean oil supplemented during the whole fermentation process was 2%, the fermentation cycle was 168 hours, the concentration of the mycelia was about 38%, the appearance was dark yellow, pH 8.4, after the pretreatment of the fermentation liquid, the titer of stallimycin was determined to be 5400 ug/mL by high performance liquid chromatography (HPLC).

Example 4: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation of Shake Flask Mycelia

[0056] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water, the loading quantity was 200 mL in a 1000 mL triangular flask. The medium was subjected to sterilization at the temperature of 121-125 C.; and at the pressure of 0.10-0.13 MPa for 30 minutes. After cooling, the medium blocks comprising spores were dug out and inoculated into the seed culture medium, then at the temperature of 292 C., they were cultured on a shaker with a rotation speed of 220 r/min for 40 hours, the concentration of mycelia was about 14%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained and had branches and segments.

(3) Preparation and Culture of Seed Tank

[0057] Formula of the medium (%): glucose 2, corn steep liquor 2.5, calcium carbonate 1.0, ammonium sulfate 0.1, potassium dihydrogen phosphate 0.01, pH6.0, it was prepared with drinking water. The loading quantity was 65 L in a 100 L seed tank, the medium was subjected to sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, after cooling, the mycelia liquid of the shake flask was inoculated, then it was cultivated at the temperature of 292 C., stirring speed 220 r/min, ventilation ratio 1:1 v/v/m for 40 hours, the concentration of the mycelia was 14%, the appearance was pale yellow, it can be observed from a microscope that the mycelia was deeply stained, thick and strong, had obvious branches and segments.

(4) Preparation and Culture of Fermentation Tank

[0058] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3.0, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, soybean oil 0.1, HABA 0.01, pH6.2, it was prepared with drinking water. The loading quantity was 700 L in a 1000 L fermentation tank, the medium was subjected to sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, after cooling, the seed liquid was inoculated with the inoculation amount of 10%, the culture temperature was 292 C., the stirring speed was 220 r/min, the ventilation ratio was 1:0.8 v/v/m. Soybean oil was added after 48 hours of culture, 1.0% was added once 24 hours, in total, the amount of soybean oil supplemented during the whole fermentation process was 4%, the fermentation cycle was 168 hours, the concentration of the mycelia was about 36%, the appearance was dark yellow, pH 7.8, after the pretreatment of the fermentation liquid, the titer of stallimycin was determined to be 5210 ug/mL by high performance liquid chromatography (HPLC).

Example 5: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Example 1

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0059] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% soybean oil was added every 24 hours (in total, the amount of soybean oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia was about 35%, the pH was 8.3, and the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 5500 ug/mL by high performance liquid chromatography (HPLC).

Example 6: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Example 1

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0060] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.05, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% soybean oil was added every 24 hours (in total, the amount of soybean oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia was about 35%, the pH was 8.0, and the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 5220 ug/mL by high performance liquid chromatography (HPLC).

Example 7: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Comparative Example 1

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Comparative Example 1

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0061] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% soybean oil was added every 24 hours (in total, the amount of soybean oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia was about 34%, the pH was 8.5, and the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 1610 ug/mL by high performance liquid chromatography (HPLC).

Example 8: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Comparative Example 2

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Comparative Example 2

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0062] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% soybean oil was added every 24 hours (in total, the amount of soybean oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia was about 35%, the pH was 8.4, and the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 2270 ug/mL by high performance liquid chromatography (HPLC).

Example 9: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Example 1

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0063] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% rapeseed oil was added every 24 hours (in total, the amount of rapeseed oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia was about 35%, the pH was 8.3, and the appearance was dark yellow 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 5400 ug/mL by high performance liquid chromatography (HPLC).

Example 10: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Example 1

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0064] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1. pH-16.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% peanut oil was added every 24 hours (in total, the amount of peanut oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia was about 38%, the pH was 8.3, and the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 5200 ug/mL by high performance liquid chromatography (HPLC).

Example 11: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Example 1

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0065] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% olive oil was added every 24 hours (in total, the amount of olive oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia was about 42%, the pH was 8.2, and the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 5230 ug/mL by high performance liquid chromatography (HPLC).

Example 12: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Example 1

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0066] Formula of the medium (%): glucose 6, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% sunflower oil was added every 24 hours (in total, the amount of sunflower oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia of the fermentation liquid was about 40%, the pH was 8.5, and the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 5210 ug/mL by high performance liquid chromatography (HPLC).

Example 13: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Example 1

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0067] Formula of the medium (%): glucose 4, dextrin 2, gluten powder 2.5, soybean meal 3, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% soybean oil was added every 24 hours (in total, the amount of soybean oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia of the fermentation liquid was about 37%, the pH was 8.2, and the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 5450 ug/mL by high performance liquid chromatography (HPLC).

Example 14: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Example 1

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0068] Formula of the medium (%): dextrin 6, gluten powder 3, soybean meal 2.5, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% soybean oil was added every 24 hours (in total, the amount of soybean oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia of the fermentation liquid was about 38%, the pH was 8.2, and the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 5330 ug/mL by high performance liquid chromatography (HPLC).

Example 15: Preparation of Stallimycin

(1) The Strain and the Preparation and Culture of Slant Spore are Seen in Example 1

(2) Preparation and Culture of Shake Flask Seed Culture Medium are Seen in Example 1

(3) Preparation and Culture of Shake Flask Fermentation Medium

[0069] Formula of the medium (%): glucose 6, gluten powder 1.5, soybean meal 4, sodium chloride 0.25, calcium carbonate 0.3, potassium dihydrogen phosphate 0.02, HABA 0.01, soybean oil 0.1. pH6.2, it was prepared with drinking water. The loading quantity was 50 mL in a 500 mL triangular flask, the medium was subjected to moist heat sterilization at the temperature of 121-125 C. and the pressure of 0.10-0.13 Mpa for 30 minutes, then the seed liquid was inoculated into the fermentation medium, the inoculation amount was 10%, the culture temperature was 292 C. After culturing on the shaker with the rotation speed of 220 r/min for 48 hours, 0.5% soybean oil was added every 24 hours (in total, the amount of soybean oil added during the whole fermentation process was 2%), they were cultivated for 168 hours and put into a bottle. The concentration of the mycelia of the fermentation liquid was about 32%, the pH was 8.4, and the appearance was dark yellow. 3 fold (volume) of 80% ethanol was added to the fermentation liquid, soaked for 30 minutes, filtered, the filtrate was collected, and the titer of stallimycin was determined to be 5390 ug/mL by high performance liquid chromatography (HPLC).