Method for improving fermentation level of avilamycin

Abstract

The invention provides a method for improving the fermentation level of avilamycin, which comprises the following steps: step 1, seed tank culture: preparing a spore solution from maturely cultured streptomyces viridochromogenes, and inoculating the spore solution into a seed tank to obtain a mature seed solution; step 2, culturing in a fermentation tank: inoculating the grown mature seed liquid into a fermentation culture medium according to an amount which is 10 percent of the volume of the inoculated mature seed liquid in the fermentation tank, and supplementing an L-valine solution, a sodium acetate solution, phytine turbid liquid and liquefied starch in the culture process, so as to obtain avilamycin fermentation liquid. According to the method, key supplements including phytin and sodium acetate are supplemented, the utilization efficiency of avilamycin A precursors and carbon sources is effectively improved in the avilamycin fermentation process through the two key supplements, the fermentation level is improved to 7329 mu g / mL from 6126 mu g / mL, the fermentation level is improved, the maximum single impurity content is effectively controlled, and finally the quality of fermentation liquor is greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial pharmaceutical manufacturing, and in particular relates to a method for improving the fermentation level of avilamycin. Background Art

[0002] Avilamycin, also known as vilamycin, amelamycin, and ferramycin, is fermented by Streptomyces viridis and is an oligosaccharide antibiotic. It can regulate bacterial metabolism in the intestines of animals, act on the ribosomes of Gram-positive bacteria and inhibit protein synthesis, and has a good inhibitory effect on Gram-positive bacteria.

[0003] Avilamicin fermentation products include more than a dozen components from Avilamicin A to Avilamicin N. The product structures and properties are similar, and separation is difficult. Avilamicin A and B are the main products in the fermentation broth, and the others are by-products or impurities. The Chinese Pharmacopoeia of Veterinary Medicine requires that the finished product use the detection method of relevant substances, and the content is calculated according to the area normalization method, requiring A% + B% ≥ 70%, A% ≥ 60%, B% ≤ 18%, and the content of other single components and impurities shall not exceed 6%.

[0004] Invention patent announcement No. CN102936608B improves the fermentation level to 6500 μg / mL by optimizing the process. It does not mention the impurity content in the fermentation broth product, and the fermentation level still has room for improvement.

[0005] The current status of avilamycin fermentation production technology in my country is still: low concentration of fermentation products and unstable quality control.

[0006] L-valine is a precursor substance for the synthesis of avilamycin A. Its content can be increased by adding it as a precursor during the fermentation process. However, a by-product will be produced during the fermentation process along with the addition of the precursor. The product tends to increase rapidly between 35-70 hours, and maintains until the tank is released after reaching the highest point. The by-product often becomes the largest single impurity in the fermentation product. Because its structure is similar to that of the main products A and B of avilamycin, and its physicochemical properties are similar, it is not easy to separate from the main product. Its high content will affect the content of impurities in the final product. Patent CN202010768287.8 solves the problem of the maximum single impurity content exceeding the requirements by adding divalent metal ions and variable temperature control. However, during the fermentation process, due to the aggregation of mycelium and the process requirements such as temperature reduction, the metabolic rate decreases, which affects the concentration of the final main product and is not conducive to the industrialization of the final process.

[0007] The main structural difference between avilamycin A and avilamycin B is that the R1 connected to the G residue structure is isobutyryl and acetyl, respectively. The R2 connected to the H residue is acetyl. The connections of other impurities are different. At present, there is little reference to the control of impurity content in avilamycin fermentation products.

[0008]

[0009] Phytin, a complex salt formed by phytate and metal calcium, magnesium and other ions, is slightly soluble in water. The hydrolyzed substances participate in metabolism, which can improve the permeability of cells, improve the efficiency of cell nutrient and oxygen transfer, and enhance the activity of enzymes, promote mass transfer and oxygen transfer between mycelium and culture medium during fermentation, and improve the conversion rate of precursors and carbon sources. Given the significant effect of Phytin in promoting mass transfer and oxygen transfer between mycelium and culture medium during fermentation, and its role as a natural complex that can promote the conversion rate of precursors and carbon sources, introducing it into the fermentation process of avilamycin has become a strategy worth exploring. Summary of the invention

[0010] The purpose of the present invention is to solve the shortcomings of the prior art. The present invention improves the fermentation level while achieving the effects of high concentration of main products and low content of by-products.

[0011] The technical solution adopted by the present invention is:

[0012] A method for improving the fermentation level of avilamycin comprises the following steps:

[0013] Step 1, seed tank culture: the mature green chromogenic Streptomyces is cultured, the spore liquid is made and inoculated into the seed tank, the temperature is controlled at 28°C-29°C, the ventilation ratio is 1.0vvm, the tank pressure is 0.03-0.04MPa, and the rotation speed is 260-300rpm, and the culture is carried out for 40-44h to obtain a mature seed liquid;

[0014] Step 2, fermentation tank culture: the grown mature seed liquid is inoculated into the fermentation medium according to 10% of the volume of the fermentation tank after filling, the ventilation ratio is 1.0vvm, the tank pressure is 0.03MPa-0.04MPa, the rotation speed is 300-500rpm, the culture temperature is 28-29°C and the culture is cultured for 240h. During the culture process, L-valine solution, sodium acetate solution, phytin suspension and liquefied starch are added to obtain avilamycin fermentation liquid.

[0015] Preferably, in step 2, when fermentation is continued for 14h-18h, 0.25% of the mass of L-valine is added to the fermentation liquid, and when fermentation is continued for 40h, 64h, 88h and 156h, 0.1% of the mass of L-valine is added to each fermentation liquid; the L-valine is prepared into an L-valine aqueous solution with a concentration (W / V) of 7% after inactivation, and the actual added mass is the mass after dryness.

[0016] Preferably, in step 2, the fermentation is continued for 30-34 hours, and the supplementary phytin suspension is added, and the first supplementary dry product amount / culture medium volume is 0.03-0.05 g / L, and then 1 / 10 of the first supplementary amount is added at 64 hours, 88 hours, and 156 hours respectively.

[0017] Preferably, in step 2, the fermentation is continued for 30-34 hours, and the feed sodium acetate solution is added, the first addition amount of dry product / culture medium volume is 0.04 g-0.06 g / L, and after the first addition, the dry sodium acetate / fermentation liquid volume is added at a constant rate of 0.04 g-0.06 g / L every 24 hours until 230 hours.

[0018] Preferably, in step 2, liquefied corn starch with a concentration of 50% (W / V) is added after 40 hours of fermentation, 20 g / L-28 g / L is added every 24 hours, and the addition is stopped after 230 hours.

[0019] Preferably, in the step 1, the formula ratio of the seed tank culture medium is calculated in g / 100mL: glucose 0.5-1.0, soluble starch 2.0-3.0, soybean cake powder 1.0-2.0, yeast powder 0.2-0.3, calcium chloride 1.8-2.2, calcium carbonate 0.1-0.2, the balance is water, sodium hydroxide is added before sterilization to adjust the pH to 7.0-7.2, and the seed culture medium is inoculated with spores of 0.1-0.30 billion spores / 20L.

[0020] Preferably, in the step 2, the formula ratio of the fermentation tank culture medium is calculated in g / 100mL: glucose 0.5-1.5, soluble starch 4.0-6.0, soybean cake powder 0.5-1.5, calcium chloride 0.1-0.3, calcium carbonate 0.4-0.6, sodium chloride 0.08-0.12, sodium nitrate 0.4-0.8, the balance of the culture medium is water, and sodium hydroxide is added before sterilization to adjust the pH to 7.2-7.4.

[0021] The beneficial effects obtained by the present invention are as follows: by adding key supplementary materials, phytin and sodium acetate, the two key supplementary materials effectively improve the utilization efficiency of the avilamycin A precursor and the carbon source during the avilamycin fermentation process, and the fermentation level is increased from 6126 μg / mL to 7329 μg / mL, which not only improves the fermentation level, but also effectively controls the maximum single impurity content, and finally the quality of the fermentation liquid is greatly improved.

[0022] After the present invention adds the phytin suspension for 30h-34h, the mycelium continues to grow and stretches without forming a mycelium ball, so that the mycelium can promote mass transfer and oxygen transfer between the mycelium and the environment, and improve the utilization rate of the precursor; the continuous addition of sodium acetate after 30h-34h provides precursor substances for the main product of avilamycin, suppresses the concentration of precursor substances of byproducts in the fermentation process, and finally effectively improves the concentration and content of the main fermentation product. Not only solves the problem of mycelium aggregation and insufficient mass transfer and oxygen transfer, but also provides strong support for improving the overall quality of the fermentation liquid by improving the conversion rate of precursors and carbon sources. It provides a new idea for the improvement of the fermentation production technology of avilamycin, is expected to promote the further development of this field, and lays a foundation for the stability of the quality of subsequent process products and the industrialization of products.

[0023] Sodium acetate, on the one hand, can be converted into acetyl-CoA by acetyl-CoA synthetase synthesized in the bacteria itself and participate in metabolism; on the other hand, it can also participate in the synthesis of the target product as a precursor of the main fermentation product. After entering the cell, its relatively high concentration inhibits the synthesis of other by-products, thus avoiding the large-scale synthesis of impurities from the source. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0025] The content and potency of avilamycin A, avilamycin B and other related substances in the fermentation broth were detected by HPLC.

[0026] Comparative Example 1

[0027] Step 1, seed tank culture: the mature green chromogenic Streptomyces was cultured, the spore liquid was made and inoculated into the seed tank, the temperature was controlled at 28°C, the ventilation ratio was 1.0vvm, the tank pressure was 0.03MPa, and the rotation speed was 260rpm. The temperature was cultured to 40 to obtain a mature seed liquid;

[0028] Step 2, fermentation tank culture: the grown seed liquid is inoculated into the fermentation medium according to 10% of the volume of the fermentation tank after filling, the ventilation ratio is 1.0vvm, the tank pressure is 0.03MPaMPa, the rotation speed is 300rpm, the culture temperature is 28°C and cultured for 240h, L-valine and liquefied starch are added during the culture process to obtain avilamycin fermentation liquid.

[0029] In the step 1, the formula ratio of the seed tank culture medium is calculated in g / 100mL: 0.5 glucose, 2.0 soluble starch, 1.0 soybean cake powder, 0.2 yeast powder, 1.8 calcium chloride, 0.1 calcium carbonate, and the balance is water. Sodium hydroxide is added before sterilization to adjust the pH to 7.0, and the seed culture medium is inoculated with 0.1-0.3 billion spores / 20L.

[0030] In the step 2, the formula ratio of the fermentation tank culture medium is calculated in g / 100mL: 0.5 glucose, 4.0 soluble starch, 0.5 soybean cake powder, 0.1 calcium chloride, 0.4 calcium carbonate, 0.08 sodium chloride and 0.4 sodium nitrate, the balance of the culture medium is water, and sodium hydroxide is added before sterilization to adjust the pH to 7.2.

[0031] In the step 2, when the fermentation is continued for 14 hours, 0.25% of the mass of L-valine is added to the fermentation liquid; when the fermentation is continued for 40 hours, 64 hours, 88 hours and 156 hours, 0.1% of the mass of L-valine is added to the fermentation liquid respectively; the L-valine is prepared into an L-valine aqueous solution, and the concentration (W / V) after inactivation is 7%, and the actual added mass is the mass after drying.

[0032] In the step 2, liquefied corn starch with a concentration of 50% (W / V) is added after 40 hours of fermentation, 20 g / L is added every 24 hours, and the addition is stopped after 230 hours.

[0033] Table 1 Comparative Example Fermentation Liquid Test Data

[0034]

[0035] Comparative Example 2

[0036] Step 1, seed tank culture: the mature green chromogenic Streptomyces was cultured, the spore liquid was made and inoculated into the seed tank, and the temperature was controlled at 28.5°C, the ventilation ratio was 1.0vvm, the tank pressure was 0.035MPa, and the rotation speed was 280rpm. Under the conditions, the culture was carried out for 42 hours to obtain a mature seed liquid;

[0037] Step 2, fermentation tank culture: the grown seed liquid is inoculated into the fermentation medium according to 10% of the volume of the fermentation tank after filling, the ventilation ratio is 1.0vvm, the tank pressure is 0.035MPa, the rotation speed is 400rpm, the culture temperature is 28.5°C and the culture is cultured for 240h. During the culture process, L-valine solution and liquefied starch are added to obtain avilamycin fermentation liquid.

[0038] In the step 1, the formula ratio of the seed tank culture medium is calculated in g / 100mL: 0.75 glucose, 2.5 soluble starch, 1.5 soybean cake powder, 0.25 yeast powder, 2.0 calcium chloride, 0.15 calcium carbonate, and the balance is water. Sodium hydroxide is added before sterilization to adjust the pH to 7.1, and the seed culture medium is inoculated with 0.1-0.3 billion spores / 20L.

[0039] In the step 2, the fermentation tank culture medium formula ratio is calculated in g / 100mL: glucose 1.0, soluble starch 5.0, soybean cake powder 1.0, calcium chloride 0.2, calcium carbonate 0.5, sodium chloride 0.10, sodium nitrate 0.6, the balance of the culture medium is water, and sodium hydroxide is added before sterilization to adjust the pH to 7.3

[0040] In the step 2, when the fermentation is continued for 16 hours, 0.25% of the mass of L-valine is added to the fermentation liquid, and when the fermentation is continued for 40 hours, 64 hours, 88 hours and 156 hours, 0.1% of the mass of L-valine is added to the fermentation liquid respectively; the L-valine is prepared into an L-valine aqueous solution, and the concentration (W / V) after inactivation is 7%, and the actual added mass is the mass after drying.

[0041] In the step 2, liquefied corn starch with a concentration of 50% (W / V) is added after 40 hours of fermentation, 24 g / L is added every 24 hours, and the addition is stopped after 230 hours.

[0042] Table 2 Comparative Example Fermentation Liquid Test Data

[0043]

[0044] Comparative Example 3

[0045] Step 1, seed tank culture: the mature green chromogenic Streptomyces was cultured, the spore liquid was made and inoculated into the seed tank, the temperature was controlled at 29°C, the ventilation ratio was 1.0vvm, the tank pressure was 0.04MPa, and the rotation speed was 300rpm, and the culture was carried out for 44 hours to obtain the mature seed liquid;

[0046] Step 2, fermentation tank culture: the grown seed liquid is inoculated into the fermentation medium according to 10% of the volume of the fermentation tank after filling, the ventilation ratio is 1.0vvm, the tank pressure is 0.04MPa, the rotation speed is 500rpm, the culture temperature is 29°C and the culture is cultured for 240h. During the culture process, liquefied starch is added to obtain avilamycin fermentation liquid.

[0047] In the step 1, the formula ratio of the seed tank culture medium is calculated in g / 100 mL: 1.0 glucose, 3.0 soluble starch, 2.0 soybean cake powder, 0.3 yeast powder, 2.2 calcium chloride, 0.2 calcium carbonate, and the balance is water. Sodium hydroxide is added before sterilization to adjust the pH to 7.2, and the seed culture medium is inoculated with 0.1-0.3 billion spores / 20L.

[0048] In the step 2, the fermentation tank culture medium formula ratio is calculated in g / 100mL: glucose 1.5, soluble starch 6.0, soybean cake powder 1.5, calcium chloride 0.3, calcium carbonate 0.6, sodium chloride 0.12, sodium nitrate 0.8, the balance of the culture medium is water, and sodium hydroxide is added before sterilization to adjust the pH to 7.4

[0049] In the step 2, when the fermentation is continued for 14h-18h, 0.25% of the mass of L-valine is added to the fermentation liquid, and when the fermentation is continued for 40h, 64h, 88h and 156h, 0.1% of the mass of L-valine is added to the fermentation liquid respectively; the L-valine is prepared into an L-valine aqueous solution, and the concentration (W / V) after inactivation is 7%, and the actual added mass is the mass after drying.

[0050] In the step 2, liquefied corn starch with a concentration of 50% (W / V) is added after 40 hours of fermentation, 28 g / L is added every 24 hours, and the addition is stopped after 230 hours.

[0051] Table 3 Comparative Example 3 Fermentation Liquid Test Data

[0052]

[0053] Example 1

[0054] Step 1, seed tank culture: the mature green chromogenic Streptomyces is cultured, the spore liquid is made and inoculated into the seed tank, the temperature is controlled at 28°C-29°C, the ventilation ratio is 1.0vvm, the tank pressure is 0.03MPa, and the rotation speed is 260rpm. Under the conditions, the spore liquid is cultured for 40 hours to obtain a mature seed liquid;

[0055] Step 2, fermentation tank culture: the grown seed liquid is inoculated into the fermentation medium according to 10% of the volume of the fermentation tank after filling, the ventilation ratio is 1.0vvm, the tank pressure is 0.03MPa, the rotation speed is 300rpm, the culture temperature is 28°C and the culture is cultured for 240h. During the culture process, L-valine solution, sodium acetate solution, phytin suspension and liquefied starch are added to obtain avilamycin fermentation liquid.

[0056] In the step 1, the formula ratio of the seed tank culture medium is calculated in g / 100mL: 0.5 glucose, 2.0 soluble starch, 1.0 soybean cake powder, 0.2 yeast powder, 1.8 calcium chloride, 0.1 calcium carbonate, and the balance is water. Sodium hydroxide is added before sterilization to adjust the pH to 7.0, and the seed culture medium is inoculated with 0.1-0.3 billion spores / 20L.

[0057] In the step 2, the fermentation tank culture medium formula ratio is calculated in g / 100mL: glucose 0.5, soluble starch 4.0, soybean cake powder 0.5, calcium chloride 0.1, calcium carbonate 0.4, sodium chloride 0.08, sodium nitrate 0.4, the balance of the culture medium is water, and sodium hydroxide is added before sterilization to adjust the pH to 7.2

[0058] In the step 2, when the fermentation is continued for 14h-18h, 0.25% of the mass of L-valine is added to the fermentation liquid, and when the fermentation is continued for 40h, 64h, 88h and 156h, 0.1% of the mass of L-valine is added to the fermentation liquid respectively; the L-valine is prepared into an L-valine aqueous solution, and the concentration (W / V) after inactivation is 7%, and the actual added mass is the mass after drying.

[0059] In the step 2, after fermentation for 30 hours, the key supplementary material, the phytin suspension, is added, and the first supplementary dry product amount / culture medium volume is 0.03 g / L, and then 1 / 10 of the first supplementary amount is added at 64 hours, 88 hours, and 156 hours respectively.

[0060] In the step 2, the key feed sodium acetate solution is added after fermentation for 30 hours, and the first addition amount is 0.04 g / L of dry product / culture medium volume. After the first addition, the sodium acetate dry product / fermentation liquid volume is added at a constant rate of 0.04 g / L every 24 hours until 230 hours.

[0061] In the step 2, liquefied corn starch with a concentration of 50% (W / V) is added after 40 hours of fermentation, 20 g / L is added every 24 hours, and the addition is stopped after 230 hours.

[0062] Table 4 Example 1 Fermentation liquid test data

[0063]

[0064] Example 2

[0065] Step 1, seed tank culture: the mature green chromogenic Streptomyces was cultured, the spore liquid was made and inoculated into the seed tank, and the temperature was controlled at 28.5°C, the ventilation ratio was 1.0vvm, the tank pressure was 0.035MPa, and the rotation speed was 280rpm. Under the conditions, the culture was carried out for 42 hours to obtain a mature seed liquid;

[0066] Step 2, fermentation tank culture: the grown seed liquid is inoculated into the fermentation medium according to 10% of the volume of the fermentation tank after connection, the ventilation ratio is 1.0vvm, the tank pressure is 0.035MPa, the rotation speed is 400rpm, the culture temperature is 28.5°C and the culture is cultured for 240h. During the culture process, L-valine solution, sodium acetate solution, phytin suspension and liquefied starch are added to obtain avilamycin fermentation liquid.

[0067] In the step 1, the formula ratio of the seed tank culture medium is calculated in g / 100mL: 0.75 glucose, 2.5 soluble starch, 1.5 soybean cake powder, 0.25 yeast powder, 2.0 calcium chloride, 0.15 calcium carbonate, and the balance is water. Sodium hydroxide is added before sterilization to adjust the pH to 7.1, and the seed culture medium is inoculated with 0.1-0.3 billion spores / 20L.

[0068] In the step 2, the fermentation tank culture medium formula ratio is calculated in g / 100mL: glucose 1.0, soluble starch 5.0, soybean cake powder 1.0, calcium chloride 0.2, calcium carbonate 0.5, sodium chloride 0.10 0.6, the balance of the culture medium is water, and sodium hydroxide is added before sterilization to adjust the pH to 7.3

[0069] In the step 2, when the fermentation is continued for 16 hours, 0.25% of the mass of L-valine is added to the fermentation liquid, and when the fermentation is continued for 40 hours, 64 hours, 88 hours and 156 hours, 0.1% of the mass of L-valine is added to the fermentation liquid respectively; the L-valine is prepared into an L-valine aqueous solution, and the concentration (W / V) after inactivation is 7%, and the actual added mass is the mass after drying.

[0070] In the step 2, after fermentation for 32 hours, the key supplement of fitin suspension is added, and the first supplement of dry product amount / culture medium volume is 0.04g / L, and then 1 / 10 of the first supplement amount is added at 64h, 88h, and 156h respectively.

[0071] In the step 2, the key feed sodium acetate solution is added after fermentation for 32 hours, and the first addition amount is 0.05 g / L of dry product / culture medium volume. After the first addition, the sodium acetate dry product / fermentation liquid volume is added at a constant rate of 0.05 g / L every 24 hours until 230 hours.

[0072] In the step 2, liquefied corn starch with a concentration of 50% (W / V) is added after 40 hours of fermentation, 24 g / L is added every 24 hours, and the addition is stopped after 230 hours.

[0073] Table 5 Example 2 Fermentation Liquid Detection Data

[0074]

[0075] Example 3

[0076] Step 1, seed tank culture: the mature green chromogenic Streptomyces was cultured, the spore liquid was made and inoculated into the seed tank, the temperature was controlled at 29°C, the ventilation ratio was 1.0vvm, the tank pressure was 0.04MPa, and the rotation speed was 300rpm, and the culture was carried out for 44 hours to obtain the mature seed liquid;

[0077] Step 2, fermentation tank culture: the grown seed liquid is inoculated into the fermentation medium according to 10% of the volume of the fermentation tank after filling, the ventilation ratio is 1.0vvm, the tank pressure is 0.04MPa, the rotation speed is 500rpm, the culture temperature is 29°C and the culture is cultured for 240h. During the culture process, L-valine solution, sodium acetate solution, phytin suspension and liquefied starch are added to obtain avilamycin fermentation liquid.

[0078] In the step 1, the formula ratio of the seed tank culture medium is calculated in g / 100 mL: 1.0 glucose, 3.0 soluble starch, 2.0 soybean cake powder, 0.3 yeast powder, 2.2 calcium chloride, 0.2 calcium carbonate, and the balance is water. Sodium hydroxide is added before sterilization to adjust the pH to 7.2, and the seed culture medium is inoculated with 0.1-0.3 billion spores / 20L.

[0079] In the step 2, the fermentation tank culture medium formula ratio is calculated in g / 100mL: glucose 1.5, soluble starch 6.0, soybean cake powder 1.5, calcium chloride 0.3, calcium carbonate 0.6, sodium chloride 0.12, sodium nitrate 0.8, the balance of the culture medium is water, and sodium hydroxide is added before sterilization to adjust the pH to 7.4

[0080] In the step 2, when the fermentation is continued for 18 hours, 0.25% of the mass of L-valine is added to the fermentation liquid, and when the fermentation is continued for 40 hours, 64 hours, 88 hours and 156 hours, 0.1% of the mass of L-valine is added to the fermentation liquid respectively; the L-valine is prepared into an L-valine aqueous solution, and the concentration (W / V) after inactivation is 7%, and the actual added mass is the mass after drying.

[0081] In the step 2, after fermentation for 34 hours, the key supplementary material, the phytin suspension, is added, and the amount of dry product added for the first time / the volume of the culture medium is 0.03-0.05 g / L, and then 1 / 10 of the first supplementary amount is added at 64 hours, 88 hours, and 156 hours respectively.

[0082] In the step 2, the key feed sodium acetate solution is added after fermentation for 34 hours, and the first addition amount is 0.06 g / L of dry product / culture medium volume. After the first addition, the sodium acetate dry product / fermentation liquid volume is added at a constant rate of 0.06 g / L every 24 hours until 230 hours.

[0083] In the step 2, liquefied corn starch with a concentration of 50% (W / V) is added after 40 hours of fermentation, 28 g / L is added every 24 hours, and the addition is stopped after 230 hours.

[0084] Table 6 Example 3 Fermentation liquid test data

[0085]

[0086] Example 4

[0087] Step 1, seed tank culture: the mature green chromogenic Streptomyces was cultured, the spore liquid was made and inoculated into the seed tank, and the temperature was controlled at 28.5°C, the ventilation ratio was 1.0vvm, the tank pressure was 0.035MPa, and the rotation speed was 280rpm. Under the conditions, the culture was carried out for 42 hours to obtain a mature seed liquid;

[0088] Step 2, fermentation tank culture: the grown seed liquid is inoculated into the fermentation medium according to 10% of the volume of the fermentation tank after connection, the ventilation ratio is 1.0vvm, the tank pressure is 0.035MPa, the rotation speed is 400rpm, the culture temperature is 28.5°C and the culture is cultured for 240h. During the culture process, L-valine solution, sodium acetate solution, phytin suspension and liquefied starch are added to obtain avilamycin fermentation liquid.

[0089] In the step 1, the formula ratio of the seed tank culture medium is calculated in g / 100mL: 0.75 glucose, 2.5 soluble starch, 1.5 soybean cake powder, 0.25 yeast powder, 2.0 calcium chloride, 0.15 calcium carbonate, and the balance is water. Sodium hydroxide is added before sterilization to adjust the pH to 7.1, and the seed culture medium is inoculated with 0.1-0.3 billion spores / 20L.

[0090] In the step 2, the fermentation tank culture medium formula ratio is calculated in g / 100mL: glucose 1.0, soluble starch 5.0, soybean cake powder 1.0, calcium chloride 0.2, calcium carbonate 0.5, sodium chloride 0.10 0.6, the balance of the culture medium is water, and sodium hydroxide is added before sterilization to adjust the pH to 7.3

[0091] In the step 2, when the fermentation is continued for 16 hours, 0.25% of the mass of L-valine is added to the fermentation liquid, and when the fermentation is continued for 40 hours, 64 hours, 88 hours and 156 hours, 0.1% of the mass of L-valine is added to the fermentation liquid respectively; the L-valine is prepared into an L-valine aqueous solution, and the concentration (W / V) after inactivation is 7%, and the actual added mass is the mass after drying.

[0092] In the step 2, after fermentation for 31 hours, the key supplementary material, the phytin suspension, is added, and the first supplementary dry product amount / culture medium volume is 0.035 g / L, and then 1 / 10 of the first supplementary amount is added at 64 hours, 88 hours, and 156 hours respectively.

[0093] In the step 2, the key feed sodium acetate solution is added after fermentation for 31 hours, and the first addition amount is 0.045 g / L of dry product / culture medium volume. After the first addition, the sodium acetate dry product / fermentation liquid volume is added at a constant rate of 0.045 g / L every 24 hours until 230 hours.

[0094] In the step 2, liquefied corn starch with a concentration of 50% (W / V) is added after 40 hours of fermentation, 22 g / L is added every 24 hours, and the addition is stopped after 230 hours.

[0095] Table 7 Example 4 Fermentation Liquid Detection Data

[0096]

[0097] Example 5

[0098] Step 1, seed tank culture: the mature green chromogenic Streptomyces was cultured, the spore liquid was made and inoculated into the seed tank, and the temperature was controlled at 28.5°C, the ventilation ratio was 1.0vvm, the tank pressure was 0.035MPa, and the rotation speed was 280rpm. Under the conditions, the culture was carried out for 42 hours to obtain a mature seed liquid;

[0099] Step 2, fermentation tank culture: the grown seed liquid is inoculated into the fermentation medium according to 10% of the volume of the fermentation tank after connection, the ventilation ratio is 1.0vvm, the tank pressure is 0.035MPa, the rotation speed is 400rpm, the culture temperature is 28.5°C and the culture is cultured for 240h. During the culture process, L-valine solution, sodium acetate solution, phytin suspension and liquefied starch are added to obtain avilamycin fermentation liquid.

[0100] In the step 1, the formula ratio of the seed tank culture medium is calculated in g / 100mL: 0.75 glucose, 2.5 soluble starch, 1.5 soybean cake powder, 0.25 yeast powder, 2.0 calcium chloride, 0.15 calcium carbonate, and the balance is water. Sodium hydroxide is added before sterilization to adjust the pH to 7.1, and the seed culture medium is inoculated with 0.1-0.3 billion spores / 20L.

[0101] In the step 2, the fermentation tank culture medium formula ratio is calculated in g / 100mL: glucose 1.0, soluble starch 5.0, soybean cake powder 1.0, calcium chloride 0.2, calcium carbonate 0.5, sodium chloride 0.10 0.6, the balance of the culture medium is water, and sodium hydroxide is added before sterilization to adjust the pH to 7.3

[0102] In the step 2, when the fermentation is continued for 16 hours, 0.25% of the mass of L-valine is added to the fermentation liquid, and when the fermentation is continued for 40 hours, 64 hours, 88 hours and 156 hours, 0.1% of the mass of L-valine is added to the fermentation liquid respectively; the L-valine is prepared into an L-valine aqueous solution, and the concentration (W / V) after inactivation is 7%, and the actual added mass is the mass after drying.

[0103] In the step 2, after fermentation for 33 hours, the key supplementary material, the phytin suspension, is added, and the first supplementary dry product amount / culture medium volume is 0.045 g / L, and then 1 / 10 of the first supplementary amount is added at 64 hours, 88 hours, and 156 hours respectively.

[0104] In the step 2, the key feed sodium acetate solution is added after fermentation for 33 hours, and the first addition amount is 0.055 g / L of dry product / culture medium volume. After the first addition, the sodium acetate dry product / fermentation liquid volume is added at a constant rate of 0.055 g / L every 24 hours until 230 hours.

[0105] In the step 2, liquefied corn starch with a concentration of 50% (W / V) is added after 40 hours of fermentation, 24 g / L is added every 24 hours, and the addition is stopped after 230 hours.

[0106] Table 8 Example 5 Fermentation Liquid Detection Data

[0107]

[0108] It can be seen from the above control examples and embodiments that the present invention effectively improves the utilization efficiency of avilamycin A precursor and carbon source by adding key supplements, phytin and sodium acetate, during the fermentation process of avilamycin, and increases the fermentation level from 6126 μg / mL to 7329 μg / mL, which not only improves the fermentation level, but also effectively controls the maximum single impurity content, and finally greatly improves the quality of the fermentation liquid.

[0109] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for improving the fermentation level of avilamycin, characterized in that: The following steps are involved: Step 1, seed tank culture: the mature green chromogenic Streptomyces is cultured, the spore liquid is made and inoculated into the seed tank, the temperature is controlled at 28°C-29°C, the ventilation ratio is 1.0vvm, the tank pressure is 0.03-0.04MPa, and the rotation speed is 260-300rpm, and the culture is carried out for 40-44h to obtain a mature seed liquid; Step 2, fermentation tank culture: the grown mature seed liquid is inoculated into the fermentation medium according to 10% of the volume of the fermentation tank after filling, the ventilation ratio is 1.0vvm, the tank pressure is 0.03MPa-0.04MPa, the rotation speed is 300-500rpm, the culture temperature is 28-29°C and the culture is cultured for 240h. During the culture process, L-valine solution, sodium acetate solution, phytin suspension and liquefied starch are added to obtain avilamycin fermentation liquid.

2. A method for improving the fermentation level of avilamycin according to claim 1, characterized in that: In the step 2, when the fermentation is continued for 14h-18h, 0.25% of the mass of L-valine is added to the fermentation liquid, and when the fermentation is continued for 40h, 64h, 88h and 156h, 0.1% of the mass of L-valine is added to the fermentation liquid respectively; the L-valine is prepared into an L-valine aqueous solution, and the concentration (W / V) after inactivation is 7%, and the actual added mass is the mass after drying.

3. A method for improving the fermentation level of avilamycin according to claim 1, characterized in that: In the step 2, after fermentation for 30-34 hours, the supplementary phytin suspension is added, and the amount of dry product added for the first time / the volume of the culture medium is 0.03-0.05 g / L, and then 1 / 10 of the first supplementary amount is added at 64 hours, 88 hours, and 156 hours respectively.

4. A method for improving the fermentation level of avilamycin according to claim 1, characterized in that: In the step 2, the sodium acetate solution is added after fermentation for 30-34 hours, and the first addition amount is 0.04g-0.06g / L of dry product / culture medium volume. After the first addition, the sodium acetate dry product / fermentation liquid volume is added at a constant rate of 0.04g-0.06g / L every 24 hours until 230 hours.

5. A method for improving the fermentation level of avilamycin according to claim 1, characterized in that: In the step 2, liquefied corn starch with a concentration of 50% (W / V) is added after 40 hours of fermentation, 20 g / L-28 g / L is added every 24 hours, and the addition is stopped after 230 hours.

6. A method for improving the fermentation level of avilamycin according to claim 1, characterized in that: In the step 1, the formula ratio of the seed tank culture medium is calculated in g / 100mL: 0.5-1.0 glucose, 2.0-3.0 soluble starch, 1.0-2.0 soybean cake powder, 0.2-0.3 yeast powder, 1.8-2.2 calcium chloride, 0.1-0.2 calcium carbonate, and the balance is water. Sodium hydroxide is added before sterilization to adjust the pH to 7.0-7.2, and the seed culture medium is inoculated with 0.1-0.30 million spores / 20L.

7. A method for improving the fermentation level of avilamycin according to claim 1, characterized in that: In the step 2, the formula ratio of the fermentation tank culture medium is calculated in g / 100mL: glucose 0.5-1.5, soluble starch 4.0-6.0, soybean cake powder 0.5-1.5, calcium chloride 0.1-0.3, calcium carbonate 0.4-0.6, sodium chloride 0.08-0.12, sodium nitrate 0.4-0.8, the balance of the culture medium is water, and sodium hydroxide is added before sterilization to adjust the pH to 7.2-7.4.

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