CN110066843B - Kitasamycin fermentation medium, kitasamycin and fermentation method thereof - Google Patents

Abstract

The invention discloses a kitasamycin fermentation medium, wherein each 1000ml of fermentation medium comprises: 3.0-10.0g of glucose, 10-25g of soybean cake powder, 10.0-25.0g of starch, 1.0-5.0g of ammonium chloride, 0.3-1.0g of monopotassium phosphate, 0.5-5.0g of magnesium sulfate, 0.03-0.1g of zinc sulfate, 1.0-5.0g of calcium carbonate and 10.0-40.0g of soybean oil, wherein the fermentation medium also comprises methyl oleate and a surfactant. According to the invention, the methyl oleate is adopted in the fermentation medium, so that the methyl oleate is easier to be hydrolyzed and metabolized, short-chain fatty acid is generated, the synthesis of macrocyclic lactone is accelerated, more acetyl coenzyme is generated, the activity of acyltransferase in the metabolic process is enhanced, the content of A5 is relatively increased, the pH value in the kitasamycin fermentation process is more stable, the content of A1 is reduced, and the defects caused by the short plates with slow soybean oil metabolism are overcome.

Description

Kitasamycin fermentation medium, kitasamycin and fermentation method thereof

Technical Field

The invention relates to the technical field of microbial fermentation, and particularly relates to a kitasamycin fermentation medium, kitasamycin and a fermentation method thereof.

Background

Kitasamycin (Kitasamycin), also called Leucomycin (Leucomycin, LM), belongs to sixteen-membered ring macrolide antibiotics, which are obtained in 1953 by fermenting, extracting and separating a Streptomyces kitasautoensis SK separated from soil of Dajubao soil in the new habitat area of Tokyo, Japan by Qin vine, and the like, and the history of more than 50 years is available so far. The kitasamycin has a structure consisting of three parts of hexahydric sugar-shake (ageeone), carbon mildew nitrogen base sugar (mye-amino) and carbon mildew sugar (Myeaeose), and can form various components with different contents due to different genetic characteristics and fermentation conditions of producing bacteria.

The kitasamycin has a structural formula:

the kitasamycin antibacterial spectrum is similar to that of erythromycin, spiramycin and tylosin, and has strong inhibition effect on gram-positive bacteria, gram-negative cocci, rickettsia, spirochete and macroviruses. Clinically, kitasamycin is mainly used for treating streptococcus, especially infection caused by hemolytic streptococcus and pneumococcus, light and moderate staphylococcus aureus infection, and patients allergic to penicillin; the effective rate of the medicine for treating bronchitis, scarlet fever, suppurative tonsillitis, septicemia and other diseases caused by streptococcus, pneumococcus and the like can reach 90 percent, and the medicine can be used for treating skin, soft tissue and respiratory tract infection, other secondary infection, osteomyelitis, diphtheria and syphilis caused by gram-positive cocci.

In recent years, because the residual quantity of kitasamycin in organisms is low, and the kitasamycin is widely applied to veterinary drugs and feed additives, the kitasamycin is used as an efficient, safe and stable drug feed additive, is mainly used for preventing and treating respiratory tract and digestive tract diseases of livestock and poultry, has a remarkable growth promotion effect on the livestock and poultry when used at a low dose, and belongs to the only macrolide antibiotics which are allowed to be used in feed for a long time in the feed drug additive use specifications issued by the ministry of agriculture.

Soybean oil is oil extracted from soybeans and is one of key raw materials for fermentation production of kitasamycin. Soybean oil is subjected to catabolism by Streptomyces beilii to produce short-chain fatty acids, which are important precursors for synthesizing kitasamycin. Modern molecular biology research shows that the kitasamycin macrolide is synthesized by condensing 5 acetic acid units, 1 propionic acid unit, 1 butyric acid unit and 1 glycolic acid unit through a polyketide (polyketide) approach similar to fatty acid synthesis, namely, lower fatty acid is connected end to end under the action of enzyme to form a fatty chain, and then an ester ring is formed under the action of cyclosynthase and modification enzyme. This indicates that an important precursor is a short chain fatty acid. In the actual production process, because the soybean oil is insoluble in water and has small specific gravity, the soybean oil can not be fully metabolized by microorganisms, the residual soybean oil influences the fermentation conditions and the fermentation level, and simultaneously brings great burden to the extraction work at the downstream of fermentation.

Methyl oleate is an unsaturated higher fatty acid ester obtained by reacting oleic acid with methanol, and the content of oleic acid in soybean oil is as high as 20-36% in the form of glyceride. Tween 80 and span 85 are nonionic surfactants, and can reduce interfacial tension of liquid and form emulsification of insoluble liquid, and Tween and span are commonly used as oil-in-water type emulsifiers, and can be emulsified, dissolved and solubilized.

In the pharmacopoeia of 2015 edition, kitasamycin A5 should be 35-70%, A4 should be 5-25%, A1 and A13 should both be 3-15%; the sum of the main components of kitasamycin A9, A8, A7, A6, A5, A4, A1, A3 and A13 must not be less than 85%. In the existing kitasamycin production, part of A1 component is higher than 15% and A5 component is lower than 35%, so that the quality of the final product can not reach the pharmacopoeia standard.

The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.

Disclosure of Invention

The invention aims to provide a kitasamycin fermentation culture medium which can stabilize the pH value in the fermentation process of kitasamycin, improve the content of A5, reduce the content of A1 and make up for the defects caused by short plates with slow soybean oil metabolism.

In order to achieve the above object, the present invention provides a kitasamycin fermentation medium, comprising per 1000ml of the fermentation medium: 3.0-10.0g of glucose, 10-25g of soybean cake powder, 10.0-25.0g of starch, 1.0-5.0g of ammonium chloride, 0.3-1.0g of monopotassium phosphate, 0.5-5.0g of magnesium sulfate, 0.03-0.1g of zinc sulfate, 1.0-5.0g of calcium carbonate and 10.0-40.0g of soybean oil, wherein the fermentation medium also comprises methyl oleate and a surfactant.

The culture medium adopts methyl oleate which is easier to metabolize than soybean oil and is converted into more acetyl coenzyme after decomposition, so that the acetyl coenzyme amount generated by soybean oil metabolism in the original fermentation ratio is compensated, and the content of the A5 component is increased.

In the fermentation process of kitasamycin, methyl oleate is smaller in molecular weight, is easier to be hydrolyzed and metabolized to generate short-chain fatty acid, synthesis of macrocyclic lactone is accelerated, and acetyl coenzyme is generated more at the same time, so that the activity of acyltransferase in the metabolic process is enhanced, the content of A5 is relatively increased, and the pH value is more stable in the fermentation process of kitasamycin due to the addition of methyl oleate, so that the content of A1 is reduced.

In a preferred embodiment, the amount of methyl oleate is 2.5 to 10.0 ml.

In a preferred embodiment, the surfactant is present in an amount of 0.5 to 2.5 ml.

In a preferred embodiment, the surfactant is composed of tween 80 and span 85.

In a preferred embodiment, the volume ratio of tween 80 to span 85 is 1: 1.

Surfactant Tween 80 and span 85 are used in the kitasamycin fermentation medium, and the solubilization and solubilization effects of the surfactant Tween 80 and the span 85 are mainly utilized, so that the dissolving and transferring of short-chain fatty acid are facilitated.

In a preferred embodiment, the surfactant is mixed with the methyl oleate and then mixed with other components.

Another object of the present invention is to provide a fermentation method of kitasamycin, comprising: inoculating the seed culture solution into any one of the kitasamycin fermentation culture media to carry out fermentation production of kitasamycin.

In a preferred embodiment, the method for preparing the seed culture solution comprises: adopting a 500ml triangular flask, wherein the culture medium is filled in 50ml per flask, and the total volume is 10-15 flasks, and the seed culture medium comprises the following components (calculated according to 1000 ml): 5.0-15.0g of glucose, 10-20g of soybean cake powder, 15.0-25.0g of starch, 2.0-5.0g of peptone, 2.0-5.0g of yeast powder, 3.0-8.0g of sodium chloride, 0.3-1.0g of monopotassium phosphate, 0.5-5.0g of magnesium sulfate, 2.0-5.0g of ammonium sulfate and 1.0-5.0g of calcium carbonate; wrapping the bottle mouth with eight layers of gauze, and sterilizing; inoculating mature bevel spores into a shake flask under an aseptic condition for culturing; after the culture is finished, taking 10 bottles with even wall hanging and merging the bottles in a sterile room into a 2000ml sterile inoculation bottle for later use;

preferably, the sterilization temperature is 121-123 ℃, and the sterilization time is 30 minutes;

preferably, the shaking table is used for culturing at the rotating speed of 220rpm, the temperature of 28.0 +/-1.0 ℃, the humidity of 30-60% and the culture time of 12-20 h.

In a preferred embodiment, the fermentation medium preparation method comprises: respectively putting other raw materials except the surfactant, methyl oleate, soybean oil and calcium carbonate into a batching container, adding a proper amount of water, fully mixing, adjusting the pH to 6.8-7.5 by using ammonia water, adding the soybean oil, uniformly mixing, adding the calcium carbonate, uniformly mixing and fixing the volume to obtain a fermentation culture medium except the surfactant and the methyl oleate, uniformly mixing the surfactant and the methyl oleate, mixing the mixture into the prepared fermentation culture medium, and finally fixing the volume in a fermentation tank to obtain a fermentation broth;

preferably, the prepared fermentation medium is sterilized at the temperature of 121-123 ℃, is kept for 30 minutes and is cooled to 28.0 ℃;

preferably, the fermentation time is 68-72 h;

preferably, the fermentation liquor is adjusted to pH 3.0-4.5 by oxalic acid and filtered; adjusting pH of the filtrate to 8.5-10.5 with ammonia water, and heating to 30-45 deg.C; adding butyl acetate for extraction, and performing back extraction on the extract by using a potassium dihydrogen phosphate buffer solution; adjusting pH of the obtained stripping solution to 7.5-10.0 with ammonia water, heating to 50-65 deg.C, and crystallizing for 30 min; and (5) performing centrifugal separation and drying to obtain a kitasamycin product.

Another object of the present invention is to provide kitasamycin obtainable by any one of the above fermentation processes. .

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, methyl oleate is adopted in the fermentation medium, and in the fermentation process of kitasamycin, as the molecular weight of methyl oleate is smaller, the methyl oleate is easier to be hydrolyzed and metabolized, short-chain fatty acid is generated, the synthesis of macrocyclic lactone is accelerated, more acetyl coenzyme is generated at the same time, and the activity of acyltransferase in the metabolic process is enhanced, so that the content of A5 is relatively improved, and as methyl oleate is added, the pH in the fermentation process of kitasamycin is more stable, so that the content of A1 is reduced, particularly the component A1 is reduced by 5.6% to the maximum extent, the requirement of 2015 pharmacopoeia on kitasamycin is completely met, and the defects caused by the short plate of soybean oil which is slowly metabolized.

(2) The fermentation medium adopts the methyl oleate surfactant Tween 80 and span 85, has the effects of solubilization and solubilization, and is favorable for the dissolution and transfer of short-chain fatty acids.

(3) The silk fibroin powder is not adopted in the fermentation medium, and the silk fibroin powder is a composite protein hydrolysate, so that the material source of the silk fibroin powder can cause the deviation of the amino acid components of the silk fibroin powder, and the unstable influence is caused on the fermentation process of the kitasamycin.

Detailed Description

The following detailed description is provided to facilitate a better understanding of the invention. It should be understood, however, that the scope of the present invention is not limited by the particular embodiments described.

The experimental conditions and experimental methods used in the examples are as follows:

seed culture conditions and process: taking 500ml triangular flasks, wherein the culture medium content of each flask is 50ml, and the total number is 10-15 flasks; the bottle mouth is bound by eight layers of gauze, the sterilization temperature is 121 ℃ and 123 ℃, and the sterilization time is 30 minutes; inoculating mature slant spore into shake flask under aseptic condition for culturing at shaking table rotation speed of 220rpm, temperature of 28.0 + -1.0 deg.C, humidity of 30-60%, and culturing time of 12-20 h; after the culture is finished, taking 10 bottles with uniform hanging walls, merging the bottles in a sterile room into a 2000ml sterile inoculation bottle for later use; wherein, the components of the seed culture medium (calculated according to 1000 ml): 5.0-15.0g of glucose, 10-20g of soybean cake powder, 15.0-25.0g of starch, 2.0-5.0g of peptone, 2.0-5.0g of yeast powder, 3.0-8.0g of sodium chloride, 0.3-1.0g of monopotassium phosphate, 0.5-5.0g of magnesium sulfate, 2.0-5.0g of ammonium sulfate and 1.0-5.0g of calcium carbonate.

Fermentation culture conditions and culture process: using a 15000ml fermentation tank (Shanghai Aokao bioengineering equipment Co., Ltd.), the volume of the fermentation medium is 9000ml, the dissolved oxygen is controlled to be more than 30%, the stirring speed is adjusted to be 0-800rpm according to the dissolved oxygen, the temperature is 28 +/-1.0 ℃, the air intake is 9L/min, and the culture time is 60-80 h; wherein the fermentation medium comprises components (calculated according to 1000 ml) except methyl oleate and surfactant: 3.0-10.0g of glucose, 10-25g of soybean cake powder, 10.0-25.0g of starch, 1.0-5.0g of ammonium chloride, 0.3-1.0g of monopotassium phosphate, 0.5-5.0g of magnesium sulfate, 0.03-0.1g of zinc sulfate, 1.0-5.0g of calcium carbonate and 10.0-40.0g of soybean oil; the preparation method of the fermentation medium comprises the following steps: respectively adding the other raw materials except the soybean oil and the calcium carbonate into a batching container, adding a proper amount of water, fully mixing, adjusting the pH to 6.8-7.5 by using ammonia water, then adding the soybean oil, uniformly mixing, and finally adding the calcium carbonate, uniformly mixing and fixing the volume.

Fermentation liquor extraction conditions and extraction process: adjusting pH of the fermentation liquor to 3.0-4.5 with oxalic acid, and filtering; adjusting pH of the filtrate to 8.5-10.5 with ammonia water, heating to 30-45 deg.C, and extracting with butyl acetate; back-extracting the extract by using a potassium dihydrogen phosphate buffer solution; adjusting pH of the stripping solution to 7.5-10.0 with ammonia water, heating to 50-65 deg.C, and crystallizing for 30 min; and (5) centrifugally separating the crystal liquid to obtain a kitasamycin product, and drying and detecting.

The method for detecting the components and the content of the kitasamycin adopts a high performance liquid detection method, and is shown in 'Chinese pharmacopoeia' 2015 edition.

The following examples all use the same seed culture conditions, fermentation culture conditions and broth extraction conditions.

Example 1

Adding 3.0ml of each of Tween 80 and span 85 into 60ml of methyl oleate, mixing uniformly, mixing with the prepared fermentation medium, and filling into a 15000ml fermentation tank to add drinking water to a constant volume of 9000 ml; sterilizing by using a steam curing tank at the temperature of 121-; and inoculating the prepared seed culture solution into a fermentation culture medium under the flame protection, and fermenting for 70 h. After extraction, high performance liquid detection is carried out, and the results are shown in the following table 1:

TABLE 1

Components	A5％	A4％	A1％	A13％	The sum of the main components%
						Percentage content	50.8	10.0	11.4	8.6	91.5

Example 2

Adding 4.5ml of each of Tween 80 and span 85 into 45ml of methyl oleate, mixing uniformly, mixing with the prepared fermentation medium, and filling into a 15000ml fermentation tank to add drinking water to a constant volume of 9000 ml; sterilizing by using a steam curing tank at the temperature of 121-; and inoculating the prepared seed culture solution into a fermentation culture medium under the flame protection, and fermenting for 72 hours. After extraction, high performance liquid detection is carried out, and the results are shown in the following table 2:

TABLE 2

Components	A5％	A4％	A1％	A13％	The sum of the main components%
						Percentage content	48.5	11.2	10.2	9.0	90.7

Example 3

Adding 6.0ml of each of Tween 80 and span 85 into 60ml of methyl oleate, mixing uniformly, mixing with the prepared fermentation medium, and filling into a 15000ml fermentation tank to add drinking water to a constant volume of 9000 ml; sterilizing by using a steam curing tank at the temperature of 121-; and inoculating the prepared seed culture solution into a fermentation culture medium under the flame protection, and fermenting for 68 hours. After extraction, high performance liquid chromatography detection was performed, and the results are shown in table 3 below:

TABLE 3

Components	A5％	A4％	A1％	A13％	The sum of the main components%
						Percentage content	49.3	11.4	9.5	9.6	91.2

Example 4

Adding 5.0ml of each of Tween 80 and span 85 into 45ml of methyl oleate, mixing uniformly, mixing with the prepared fermentation medium, and filling into a 15000ml fermentation tank to add drinking water to a constant volume of 9000 ml; sterilizing by using a steam curing tank at the temperature of 121-; and inoculating the prepared seed culture solution into a fermentation culture medium for fermentation for 72 hours under the flame protection. After extraction, high performance liquid chromatography detection was performed, and the results are shown in table 4 below:

TABLE 4

Components	A5％	A4％	A1％	A13％	The sum of the main components%
						Percentage content	51.0	10.6	8.9	9.5	90.8

Comparative example (methyl oleate is not contained in the fermentation Medium)

Uniformly mixing 6.0ml of each of Tween 80 and span 85, mixing the mixture into the prepared fermentation medium, and filling the mixture into a 15000ml fermentation tank to add drinking water to a constant volume of 9000 ml; sterilizing by using a steam curing tank at the temperature of 121-; and inoculating the prepared seed culture solution into a fermentation culture medium under the flame protection, and fermenting for 72 hours. After extraction, high performance liquid chromatography detection was performed, and the results are shown in table 5 below:

TABLE 5

Components	A5％	A4％	A1％	A13％	The sum of the main components%
						Percentage content	44.5	11.0	14.5	8.9	90.5

Comparing the data in table 5 with the corresponding data in tables 1-4, it can be seen that: tables 1-4, a 5% is 50.8%, 48.5%, 49.3%, 51.0%, respectively, while a 5% in table 5 is 44.5%, lower than the percentage of a5 in tables 1-4, i.e. the content of a5 in the kitasamycin product is reduced when the fermentation medium does not contain methyl oleate; the a 1% in tables 1-4 was 11.4%, 10.2%, 9.5%, 8.9%, respectively, while the a 1% in table 5 was 14.5%, which is higher than the a 1% in tables 1-4, i.e. the content of a1 in the kitasamycin product was increased when the fermentation medium did not contain methyl oleate.

In the examples 1-4, the fermentation medium is added with methyl oleate, and compared with the case of the comparative example in which the medium is not added with methyl oleate, the content of the A5 component in the kitasamycin product obtained by fermentation is improved, the content of the A1 component is reduced, particularly the A1 component is reduced by 5.6% to the maximum, and the requirements of the 2015 pharmacopoeia on kitasamycin are completely met.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims (3)

1. A kitasamycin fermentation medium comprising per 1000ml of said fermentation medium: 3.0-10.0g of glucose, 10-25g of soybean cake powder, 10.0-25.0g of starch, 1.0-5.0g of ammonium chloride, 0.3-1.0g of monopotassium phosphate, 0.5-5.0g of magnesium sulfate, 0.03-0.1g of zinc sulfate, 1.0-5.0g of calcium carbonate and 10.0-40.0g of soybean oil, and is characterized in that the fermentation medium also comprises methyl oleate and a surfactant; the content of the methyl oleate is 2.5-10.0ml, and the content of the surfactant is 0.5-2.5 ml;

the surfactant consists of tween 80 and span 85.

2. The kitasamycin fermentation medium according to claim 1, wherein the volume ratio of tween 80 and span 85 is 1: 1.

3. The kitasamycin fermentation broth of claim 1 wherein said surfactant is mixed with methyl oleate before being mixed with the other ingredients.