阅读说明：本技术 一种用于生产去甲基金霉素的发酵培养基 (Fermentation medium for producing demethylated aureomycin ) 是由 韩冰 孙翠玲 刘聪洁 王绘砖 徐珍 袁昉 于 2020-05-28 设计创作，主要内容包括：本发明属于发酵培养技术领域,具体涉及一种用于生产去甲基金霉素的发酵培养基,所述发酵培养基中含有碳源,氮源和磷源,还添加了糖蜜作为提供生物素及泛酸的来源,还添加了氯化钙替代氯化钠作为无机盐来源。本发明既能提高去甲基金霉素效价又能降低杂质去甲基四环素含量,并且高效、简单、低成本。(The invention belongs to the technical field of fermentation culture, and particularly relates to a fermentation culture medium for producing demethylated aureomycin. The invention can improve the potency of the demethylated aureomycin, reduce the content of impurity demethylated tetracycline, and has high efficiency, simplicity and low cost.)

1. A fermentation medium for producing demethylated aureomycin, said fermentation medium comprising a carbon source, a nitrogen source and a phosphorus source, characterized in that molasses is further added as a source for biotin and pantothenic acid, and calcium chloride is further added as a source of inorganic salts instead of sodium chloride.

2. The fermentation medium for producing demethylated aureomycin according to claim 1, wherein said fermentation medium comprises the following composition: 60g/L of corn starch, 0.5-1.5g/L of molasses, 35g/L of high-temperature soybean powder, 25g/L of corn oil, 15g/L of corn steep liquor, 5.5g/L of calcium carbonate, 2-4g/L of calcium chloride, 1.2g/L of ammonium sulfate and 0.15g/L of potassium dihydrogen phosphate. L-lysine salt 7g/L, a-amylase 0.05 g/L. .

3. The fermentation medium for producing demethylated aureomycin according to claim 2, wherein said fermentation medium comprises the following composition: 70g/L of corn starch, 0.5g/L of molasses, 35g/L of high-temperature soybean powder, 25g/L of corn oil, 15g/L of corn steep liquor, 5.5g/L of calcium carbonate, 2g/L of calcium chloride, 1.2g/L of ammonium sulfate and 0.15g/L of monopotassium phosphate. L-lysine salt 7g/L, a-amylase 0.05 g/L.

4. The fermentation medium for producing demethylated aureomycin according to claim 2, wherein said fermentation medium comprises the following composition: 70g/L of corn starch, 1g/L of molasses, 35g/L of high-temperature soybean powder, 25g/L of corn oil, 15g/L of corn steep liquor, 5.5g/L of calcium carbonate, 3g/L of calcium chloride, 1.2g/L of ammonium sulfate and 0.15g/L of monopotassium phosphate. L-lysine salt 7g/L, a-amylase 0.05 g/L.

5. A fermentation medium for the production of demethylated aureomycin according to claim 2, characterized in that it has the composition: 70g/L of corn starch, 1.5g/L of molasses, 35g/L of high-temperature soybean powder, 25g/L of corn oil, 15g/L of corn steep liquor, 5.5g/L of calcium carbonate, 4g/L of calcium chloride, 1.2g/L of ammonium sulfate and 0.15g/L of monopotassium phosphate. L-lysine salt 7g/L, a-amylase 0.05 g/L.

Technical Field

The invention belongs to the technical field of fermentation culture, and particularly relates to a fermentation culture medium for producing demethylated aureomycin.

Background

Demethylated aureomycin is a tetracyclic antibiotic, having the english name demethylchlortracecycline, and is characterized by stronger action strength than tetracycline and oxytetracycline and more stable than aureomycin. The mechanism of demethylated aureomycin bacteriostasis, a member of tetracyclic antibiotics, is that it can specifically bind to the A position of the 30S subunit of the bacterial ribosome, thereby preventing the connection of aminoacyl-tRNA with this position and thus delaying or disabling the synthesis of bacterial proteins. The Chinese medicinal composition is clinically used for treating pneumonia, urinary tract infection, gonorrhea, bacillary dysentery, infantile scarlet fever and the like. In addition to clinical medical use, demeclocycline is also a precursor for the synthesis of minocycline and tigecycline, and is an important member of the biological fermentation of antibiotics. The yield problem of the demethylated aureomycin is prominent, and the problem that the demethylated tetracycline impurity is high in the fermentation process of the demethylated aureomycin is prominent, but the existing fermentation culture medium at home and abroad can only take the whole aspects into consideration, and can not improve the yield of the demethylated aureomycin and reduce the demethylated tetracycline impurity. According to the report of US3050446 (1962), the addition of copper sulfate can reduce the impurity demethyltetracycline, although the content of demethyltetracycline is reduced with the increasing addition of copper sulfate, the titer of demethyltetracycline is reduced, and copper sulfate is heavy ion and has certain harm to human health. According to the report of US patent No. 3145154(1964), the addition of D-methionine to the fermentation medium of demethylated aureomycin reduces the content of nortetracycline as an impurity. When 0.5% D-methionine was added to the fermentation medium, the ratio of nortetracycline to noraureomycin as an impurity was reduced from 12% when it was not added to 3%, but the noraureomycin titer was reduced from 5360ug/ml when it was not added to 4930 ug/ml. From an economic perspective, D-methionine is more expensive than bulk feedstocks, greatly increasing the operating costs of the fermentation. Chinese patent CN201310575478.2 discloses a culture medium and a culture method for producing demethylated aureomycin, the fermentation period of the demethylated aureomycin is 180h, the fermentation titer is 13000mg/L, but the fermentation process of the demethylated aureomycin is three-stage fermentation, and the culture medium contains animal-derived raw materials such as fish oil, and the culture medium is difficult to be industrially applied according to the GMP requirements of veterinary drugs.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides the demethylated aureomycin fermentation culture medium which is high-efficiency, simple and low-cost, and can improve the titer of demethylated aureomycin and reduce the content of impurity demethylated tetracycline.

The invention also provides a fermentation medium for producing the demethylated aureomycin, which contains a carbon source, a nitrogen source and a phosphorus source, molasses is added as a source for providing biotin and pantothenic acid, and calcium chloride is added to replace sodium chloride as an inorganic salt source.

The fermentation medium comprises the following components: 60g/L of corn starch, 0.5-1.5g/L of molasses, 35g/L of high-temperature soybean powder, 25g/L of corn oil, 15g/L of corn steep liquor, 5.5g/L of calcium carbonate, 2-4g/L of calcium chloride, 1.2g/L of ammonium sulfate and 0.15g/L of potassium dihydrogen phosphate. L-lysine salt 7g/L, a-amylase 0.05 g/L. .

The fermentation medium comprises the following components: 70g/L of corn starch, 0.5g/L of molasses, 35g/L of high-temperature soybean powder, 25g/L of corn oil, 15g/L of corn steep liquor, 5.5g/L of calcium carbonate, 2g/L of calcium chloride, 1.2g/L of ammonium sulfate and 0.15g/L of monopotassium phosphate. L-lysine salt 7g/L, a-amylase 0.05 g/L.

The fermentation medium comprises the following components: 70g/L of corn starch, 1g/L of molasses, 35g/L of high-temperature soybean powder, 25g/L of corn oil, 15g/L of corn steep liquor, 5.5g/L of calcium carbonate, 3g/L of calcium chloride, 1.2g/L of ammonium sulfate and 0.15g/L of monopotassium phosphate. L-lysine salt 7g/L, a-amylase 0.05 g/L.

The fermentation medium comprises the following components: 70g/L of corn starch, 1.5g/L of molasses, 35g/L of high-temperature soybean powder, 25g/L of corn oil, 15g/L of corn steep liquor, 5.5g/L of calcium carbonate, 4g/L of calcium chloride, 1.2g/L of ammonium sulfate and 0.15g/L of monopotassium phosphate. L-lysine salt 7g/L, a-amylase 0.05 g/L.

The invention has the beneficial effects that: in the invention, molasses is added to a fermentation medium of the demethyl aureomycin for the first time to serve as a source for providing biotin and pantothenic acid, because the molasses contains a large amount of biotin and pantothenic acid. Coenzyme A carboxytransferase is the main enzyme for producing the malonic acid as a demethyl aureomycin precursor, and the coenzyme A carboxytransferase belongs to a receptor of carboxytransferase by taking acyl coenzyme A as carbon dioxide, and the main coenzyme thereof is biotin, while pantothenic acid is the main component of coenzyme A, so that the supply of a large amount of biotin and pantothenic acid can increase the contents of coenzyme A carboxytransferase and coenzyme A, thereby increasing the production of the precursor and thus increasing the content of demethyl aureomycin. In the invention, calcium chloride is used for replacing sodium chloride in the fermentation culture medium of the demethyl aureomycin for the first time and is added into the fermentation culture medium of the demethyl aureomycin, so that the titer of the demethyl aureomycin can be improved, and the content of impurity demethyl tetracycline can be reduced. Because calcium chloride is soluble salt, calcium ions can be completely dissolved in water and can be directly utilized by microorganisms. Calcium ions belong to a second messenger molecule and have important regulation effects on microorganisms, particularly streptomyces, for example, calcium ions have the effects of promoting growth of aerial hyphae and activating calmodulin on some streptomyces. The invention finds that calcium ions have important influence on the early morphological differentiation and physiological differentiation of the demethyl aureomycin producing strain streptomyces aureus. At the initial stage of culturing of the streptomyces aureus, calcium ions can promote the hypha form and the in-vivo metabolic pathway of the streptomyces aureus to be more favorable for synthesizing demethyl aureomycin at the later stage, and simultaneously the calcium ions can enhance the expression of demethyl aureomycin genes in the later stage, so that the activity and the concentration of demethyl aureomycin synthetase at the later stage are enhanced, and the titer unit of demethyl aureomycin is improved and the content of impurity demethyl tetracycline is reduced. Calcium carbonate in a fermentation medium is water-insoluble inorganic salt, and in the early fermentation process, the streptomyces aureus cannot utilize calcium ions contained in the calcium carbonate and cannot promote early morphological differentiation and physiological differentiation of the streptomyces aureus, so that the function of promoting the synthesis of late demethylaureomycin is not achieved.

Detailed Description

The present invention is described in detail below with reference to specific examples:

since the seed culture medium and the culture conditions, and the fermentation culture conditions all adopt the technology disclosed in the industry, the unified seed culture medium and culture conditions, and the culture conditions of the fermentation bottle are adopted in the following examples, so as to more intuitively show the effect of the fermentation culture medium of the invention.

The components and the proportion (g/L) of the seed culture medium are as follows: 30 parts of starch, 25 parts of high-temperature soybean powder, 10 parts of peptone, 2 parts of ammonium sulfate, 1.5 parts of dipotassium hydrogen phosphate, 10 parts of corn oil, 8 parts of calcium carbonate and 0.05g/L, PH parts of alpha-amylase.

A cell bank of streptomyces aureofaciens producing demethyl aureomycin of the company is used for carrying out sandy soil construction on an eggplant bottle inclined plane, 2cm of inclined plane spores are taken to be inoculated into a 250ml shake flask filled with 40ml of seed culture medium after the eggplant bottle inclined plane is mature, the culture temperature is 28 ℃, the rotating speed of a shaking table is 200rpm, and the culture period is 48 hours. 2ml of mature seed culture medium is inoculated into 10 250ml shake flasks filled with 40ml of fermentation medium, the culture temperature is 28 ℃, the rotation speed of a shaking table is 200rpm, and the culture period is 168 hours. After the fermentation was terminated, the contents of demethylated aureomycin and demethylated tetracycline were determined by HPLC and the data were averaged over 10 flasks.

Example 1

a, preparing a fermentation medium: 28g of corn starch, 0.2g of molasses, 14g of high-temperature soybean powder, 10g of corn oil, 6g of corn steep liquor, 2.2g of calcium carbonate, 0.8g of calcium chloride, 0.48g of ammonium sulfate, 0.06g of monopotassium phosphate, 2g of L-lysine salt and 0.002g of alpha-amylase are weighed. Gelatinizing starch, adding amylase, cooling, dissolving other components in purified water, mixing the two solutions, diluting to 400ml, adjusting pH to 5.5, packaging into 10 250ml shake flasks, sterilizing at 121 deg.C for 30min, and cooling to room temperature.

b, taking 2ml of mature seed culture solution, inoculating the mature seed culture solution into 10 250ml shake flasks filled with 40ml of fermentation medium, wherein the culture temperature is 28 ℃, the rotation speed of a shaking table is 200rpm, and the culture period is 168 hours. After the fermentation is terminated, the contents of demethylated aureomycin and demethylated tetracycline are determined by high pressure liquid chromatography, and the average value of the data of 10 fermentation bottles is taken, and the specific data is shown in the following table:

example 2

a, preparing a fermentation medium: 28g of corn starch, 0.4g of molasses, 14g of high-temperature soybean powder, 10g of corn oil, 6g of corn steep liquor, 2.2g of calcium carbonate, 1.2g of calcium chloride, 0.48g of ammonium sulfate, 0.06g of monopotassium phosphate, 2g of L-lysine salt and 0.002g of alpha-amylase are weighed. Gelatinizing starch, adding amylase, cooling, dissolving other components in purified water, mixing the two solutions, diluting to 400ml, adjusting pH to 5.5, packaging into 10 250ml shake flasks, sterilizing at 121 deg.C for 30min, and cooling to room temperature.

b, taking 2ml of mature seed culture solution, inoculating the mature seed culture solution into 10 250ml shake flasks filled with 40ml of fermentation medium, wherein the culture temperature is 28 ℃, the rotation speed of a shaking table is 200rpm, and the culture period is 168 hours. After the fermentation is terminated, the contents of demethylated aureomycin and demethylated tetracycline are determined by high pressure liquid chromatography, and the average value of the data of 10 fermentation bottles is taken, and the specific data is shown in the following table:

example 3

a, preparing a fermentation medium: 28g of corn starch, 0.6g of molasses, 14g of high-temperature soybean powder, 10g of corn oil, 6g of corn steep liquor, 2.2g of calcium carbonate, 1.6g of calcium chloride, 0.48g of ammonium sulfate, 0.06g of monopotassium phosphate, 2g of L-lysine salt and 0.002g of alpha-amylase are weighed. Gelatinizing starch, adding amylase, cooling, dissolving other components in purified water, mixing the two solutions, diluting to 400ml, adjusting pH to 5.5, packaging into 10 250ml shake flasks, sterilizing at 121 deg.C for 30min, and cooling to room temperature.

b, taking 2ml of mature seed culture solution, inoculating the mature seed culture solution into 10 250ml shake flasks filled with 40ml of fermentation medium, wherein the culture temperature is 28 ℃, the rotation speed of a shaking table is 200rpm, and the culture period is 168 hours. After the fermentation is terminated, the contents of demethylated aureomycin and demethylated tetracycline are determined by high pressure liquid chromatography, and the average value of the data of 10 fermentation bottles is taken, and the specific data is shown in the following table:

example 4

a, preparing a fermentation medium: 28g of corn starch, 14g of high-temperature bean flour, 10g of corn oil, 6g of corn steep liquor, 2.2g of calcium carbonate, 0.48g of ammonium sulfate, 0.06g of monopotassium phosphate, 2g of L-lysine salt and 0.002g of alpha-amylase are weighed. Gelatinizing starch, adding amylase, cooling, dissolving other components in purified water, mixing the two solutions, diluting to 400ml, adjusting pH to 5.5, packaging into 10 250ml shake flasks, sterilizing at 121 deg.C for 30min, and cooling to room temperature.

b, taking 2ml of mature seed culture solution, inoculating the mature seed culture solution into 10 250ml shake flasks filled with 40ml of fermentation medium, wherein the culture temperature is 28 ℃, the rotation speed of a shaking table is 200rpm, and the culture period is 168 hours. After the fermentation is terminated, the contents of demethylated aureomycin and demethylated tetracycline are determined by high pressure liquid chromatography, and the average value of the data of 10 fermentation bottles is taken, and the specific data is shown in the following table:

in the above examples 1 to 3, the fermentation broth medium of the present invention was used, and in example 4, the fermentation medium of the prior art was used, and from the data, it was clearly seen that the titer of the fermentation medium of the present invention was about 40% higher than that of the fermentation medium of the prior art, and the content of impurity demethyltetracycline was reduced to about 1.5%.