阅读说明：本技术 一种以金色链霉菌发酵生产四环素的发酵培养基及补料方法 (Fermentation medium for producing tetracycline by fermenting streptomyces aureofaciens and feeding method ) 是由 李学兵 郭海燕 孙瑞君 张志� 胡晓玲 胡钦龙 冯占梅 于 2019-11-06 设计创作，主要内容包括：本发明涉及一种以金色链霉菌发酵生产四环素的发酵培养基及补料方法,该所述发酵培养基及补料基料均采用甲酯化油脂作为辅助碳源。本发明引入甲酯化油脂,使其与淀粉形成辅助碳源,可以有效阻止脂肪酸的合成,从而加强产物代谢的通路,从而提高发酵水平,降低淀粉的消耗。(The invention relates to a fermentation medium and a feeding method for producing tetracycline by fermenting streptomyces aureofaciens, wherein the fermentation medium and a feeding base material both adopt methyl esterified grease as an auxiliary carbon source. The methyl esterified grease is introduced to form an auxiliary carbon source with starch, so that the synthesis of fatty acid can be effectively prevented, the pathway of product metabolism is enhanced, the fermentation level is improved, and the consumption of starch is reduced.)

1. A fermentation medium for producing tetracycline by fermentation of streptomyces aureofaciens is characterized in that the fermentation medium adopts methyl esterified grease as an auxiliary carbon source, the proportion of the methyl esterified grease in the fermentation culture is 0.2-0.5%, and the proportion of the methyl esterified grease to starch is 1: 8-1: 12.

2. The fermentation medium according to claim 1, wherein the fermentation medium comprises the following raw materials: 2-5% of starch, 0.2-0.5% of methyl esterified grease, 1-3% of soybean cake powder, 2.0-3.0% of corn steep liquor, 0.2-0.5% of yeast powder, 0.3-1.0% of ammonium sulfate, 0.3-0.8% of light calcium carbonate and proper amounts of trace elements and inorganic salt.

3. The culture medium according to claim 1 or 2, wherein the methyl-esterified oil is vegetable oil and methyl-esterified oil obtained by methyl esterification reaction, and the methyl ester rate of the methyl-esterified oil is more than 80%.

4. The methylated oil according to claim 3, wherein the vegetable oil is soybean oil, cotton seed oil, rapeseed oil or palm oil.

5. A method for supplementing carbon source and ammonia water to fermentation broth for producing tetracycline by using the fermentation medium is characterized in that the fermentation broth is supplemented with carbon source and ammonia water in the fermentation process;

the carbon source supplement refers to supplement of starch sugar and methyl esterified oil in the fermentation process, wherein

Starch sugar is supplemented when the total sugar of the fermentation liquor is reduced to below 2.5 percent, the total sugar is controlled to be 2.0 to 3.0 percent in the fermentation process,

supplementing methyl ester oil, feeding supplement when fermenting for 20-30 h, the supplement rate is 0.1-0.2 g/hr/L culture solution,

the ratio of the amount of the methyl esterification oil to the amount of the starch sugar in the whole process is 1: 8-1: 12, and the methyl esterification oil is stopped being supplemented 20-30 hr before the tank is placed;

and the ammonia water supplement means that ammonia water is supplemented according to the pH value of the fermentation liquor, and the pH value of the fermentation liquor in the fermentation process is controlled to be 5.7-6.0.

Technical Field

The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation medium for producing tetracycline by a streptomyces aureus fermentation method and a feeding method.

Background

The tetracycline is a tetracyclic antibiotic, and is mostly produced by a fermentation method at present, streptomyces aureofaciens is taken as a fermentation strain in the fermentation process, and the tetracycline is produced by a secondary fermentation mode, and the specific process is as follows: streptomyces aureofaciens is rejuvenated and separated, spores are cultured to prepare slant spores for later use, then the slant spores are inoculated into a seeding tank for seed amplification culture, finally the cultured seeds are inoculated into a fermentation culture medium, the primary and secondary metabolism of thalli is controlled by process material supplement, culture temperature, pH, substrate concentration and the like, and finally the thalli are synthesized into tetracycline.

In the prior art, a tetracycline fermentation medium adopts mixed nutrient substances consisting of starch, soybean cake powder, yeast powder, inorganic salt, trace elements and the like, and the fermentation process adopts mixed starch sugar consisting of starch, corn steep liquor, trace elements and the like as a supplement, and ammonia water is supplemented to control the pH value in the fermentation process. Wherein the carbon source substance in the fermentation process takes starch as the main component, and the oil carbon source is not used in the fermentation system. In the prior art, the unit consumption of starch is 9-12 kg/billion tetracycline in the tetracycline fermentation process, and the consumption is extremely high. A large amount of grain crops are consumed in the fermentation process, so that the cost of raw materials is high, a fermentation unit cannot be improved, and the carbon residue in a fermentation tank is high, so that the difficulty in subsequent waste residue treatment is increased. Therefore, the unit consumption of the tetracycline fermentation carbon source is reduced, the consumption of grain crops such as starch is reduced, and the adoption of economic crops as the carbon source and the like is the technical key of the current tetracycline fermentation development.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a fermentation culture medium for producing tetracycline by fermenting streptomyces aureofaciens, which can effectively reduce the unit consumption of starch, improve the fermentation unit and reduce the cost of raw materials.

Another object of the present invention is to provide the above feeding method for producing tetracycline by fermentation of Streptomyces aureofaciens.

According to the invention, a methylated oil system is introduced in the fermentation process, and the metabolism of a carbon source to the fat synthesis direction is reduced, so that the metabolism to the product direction is enhanced, the product synthesis efficiency of the carbon source is improved, and further the fermentation unit is improved. The specific technical scheme is as follows:

a fermentation medium for producing tetracycline by fermentation of streptomyces aureofaciens is characterized in that the fermentation medium adopts methyl esterified grease as an auxiliary carbon source, the proportion of the methyl esterified grease in the fermentation culture is 0.2-0.5%, and the proportion of the methyl esterified grease to starch is 1: 8-1: 12.

The fermentation medium comprises the following specific raw materials: 2-5% of starch, 0.2-0.5% of methyl esterified grease, 1-3% of soybean cake powder, 2.0-3.0% of corn steep liquor, 0.2-0.5% of yeast powder, 0.3-1.0% of ammonium sulfate, 0.3-0.8% of light calcium carbonate and proper amounts of trace elements and inorganic salt.

The methyl esterified oil is vegetable oil and methyl esterified oil obtained by methyl esterification reaction, and the methyl ester rate of the methyl esterified oil is more than 80 percent.

The vegetable oil is soybean oil, cotton seed oil, rapeseed oil or palm oil.

The feed supplement method for producing tetracycline by fermentation of the fermentation medium is characterized in that the carbon source and ammonia water are supplemented to the neutral fermentation liquor in the fermentation process,

the carbon source supplement refers to supplement of starch sugar and methyl esterified oil in the fermentation process, wherein

Starch sugar is supplemented when the total sugar of the fermentation liquor is reduced to below 2.5 percent, the total sugar is controlled to be 2.0 to 3.0 percent in the fermentation process,

supplementing methyl ester oil, feeding supplement when fermenting for 20-30 h, the supplement rate is 0.1-0.2 g/hr/L culture solution,

the ratio of the amount of the methyl esterification oil to the amount of the starch sugar in the whole process is 1: 8-1: 12, and the methyl esterification oil is stopped being supplemented 20-30 hr before the tank is placed;

and the ammonia water supplement means that ammonia water is supplemented according to the pH value of the fermentation liquor, and the pH value of the fermentation liquor in the fermentation process is controlled to be 5.7-6.0.

The conventional tetracycline fermentation process only supplements starch sugar and ammonia water, and the methyl esterified grease is introduced to form an auxiliary carbon source with starch, so that the synthesis of fatty acid can be effectively prevented, a product metabolism passage is enhanced, the fermentation level is improved, and the consumption of the starch is reduced. The technical advantages are as follows:

1. the methyl esterification oil synthesized by degrading natural oil is used as a fermentation culture medium and a supplementary nutrient of the tetracycline generating bacteria, and because the oil forms fatty acid methyl ester after methyl esterification, the fatty chain is shortened and is easy to decompose and utilize by the tetracycline generating bacteria, partial fatty acid and methyl are provided for fermentation metabolism, and the partial substances can be used as precursor substances of the fermentation metabolism, thereby being beneficial to the synthesis of the tetracycline.

2. According to the invention, the methyl esterification oil is added into the tetracycline fermentation base material, so that the induction utilization of bacteria to the methyl esterification oil can be promoted, the establishment of an enzyme system is started, an initial enzyme system is provided for process feeding, the effective utilization of the methyl esterification oil after the methyl esterification oil is supplemented can be ensured, the accumulation is avoided, the starch feeding amount is reduced, the viscosity of the fermentation solution is reduced, the oxygen supply is improved, and the good growth of the bacteria in the fermentation process is ensured.

3. According to the invention, the methyl esterification oil is fed during fermentation for 20-30 h, and is fed into the secondary metabolism early stage after the tetracycline bacteria mass is formed, the bacteria are formed by using the methyl esterification oil in a basic culture medium to induce an enzyme system, and a sustainable utilization process is formed with the fed methyl esterification oil, so that the flux of a carbon source to a fat synthesis path is prevented or reduced, the conversion of the bacteria to the direction of synthesizing tetracycline is facilitated, and the improvement of the fermentation target product tetracycline is achieved.

4. After the methyl esterification oil is used for tetracycline fermentation, the use of grain crops can be saved, the methyl esterification oil can be obtained by carrying out methyl esterification on economic crops such as soybean oil, rapeseed oil, cottonseed oil, palm oil and the like, the process scheme is mature, the byproduct glycerol can be separated by the methyl esterification process, the oil cost is reduced, and the raw material cost of the tetracycline fermentation is reduced.

5. By adopting the method, the tetracycline fermentation tank-placing index can be increased to 2.0-2.1 from 1.8-1.9, the tank-placing titer can reach 33000-34000 u/ml, the fermentation period is shortened by more than 10 hours, the unit consumption of starch is reduced by 20-30%, the total sugar in the tank-placing fermentation liquor is reduced by more than 20%, the cost of fermentation raw materials can be reduced by 10-20%, the viscosity of the fermentation liquor is reduced in the fermentation process, the oxygen supply capacity in the fermentation process is effectively improved, the energy consumption is reduced, the quality of the tank-placing fermentation liquor is improved, and the filtration speed of the fermentation liquor is improved.

Detailed Description

The following describes in detail embodiments of the present invention with reference to examples. It should be understood that the examples are intended to illustrate the invention and are not intended to limit the invention. The scope and core content of the invention are to be determined by the claims.

In the following examples, the methylated oil was obtained by: the method is purchased in the market, natural oil and methanol can be subjected to methyl esterification reaction under the catalysis of alkali, and glycerol is separated to obtain methyl esterified oil, wherein the methyl ester rate is more than 80%. The natural oil is natural vegetable oil, including soybean oil, cotton seed oil, rapeseed oil or palm oil.

The following examples are run into the tank in phase with the conventional fermentation protocol and compare the fermentation level changes. The conventional tetracycline fermentation process is as follows:

inoculating the slant spores into a sterilized seed culture medium, and performing seed culture.

Seed culture medium: 3-4% of starch, 2-3% of soybean cake powder, 0.3-1.0% of yeast powder, 0.2-1.0% of corn steep liquor, 0.1-0.7% of ammonium sulfate, 0.1-0.5% of light calcium carbonate and proper amounts of trace elements and inorganic salt.

Seed culture conditions: culturing for 20-28 hr under stirring at 30-35 deg.C and ventilation rate of 1: 0.8-1.2 v/v/min to obtain seed liquid.

And inoculating the cultured seed solution into a sterilized fermentation culture medium for fermentation culture, wherein the inoculation amount is 10-20%. The culture period is 170-180 hours.

Fermentation medium: 7-10% of starch, 1.0-3.0% of soybean cake powder, 0.2-0.7% of yeast powder, 2.0-3.0% of corn steep liquor, 0.3-1.0% of ammonium sulfate, 0.3-0.8% of light calcium carbonate and proper amounts of trace elements and inorganic salt.

And (3) fermentation culture process: adding starch sugar according to the total sugar level, and controlling the total sugar to be 2.0-4.0%; adding ammonia water according to the pH value, and controlling the pH value to be 5.8-6.0. The culture temperature is 29-33 ℃, the air is introduced in the culture medium according to the ratio of 1: 1.1-1.4 v/v/min, and the culture medium is stirred.

Culturing for about 170-180 hours, putting into a tank, wherein the tank-putting titer is 30000-32000 mu/ml, the total sugar in the tank is 2.0-2.5%, the fermentation index is 1.8-1.9, and the unit consumption of starch is 10-12 kg/billion tetracycline.

Example 1

The seed culture mode is not changed, and the fermentation culture condition is not changed.

The soybean oil methyl ester oil is selected as the fermentation culture medium and the supplementary material, and the methyl ester rate is 81.2 percent.

The fermentation medium comprises the following components: 2.3 percent of starch, 0.2 percent of methyl esterified grease, 3 percent of soybean cake powder, 0.25 percent of yeast powder, 2.5 percent of corn steep liquor, 0.65 percent of ammonium sulfate, 0.45 percent of light calcium carbonate and proper amount of trace elements and inorganic salt, wherein the ratio of methyl esterified oil to starch in the base material is 1: 8.7.

And (3) adding starch sugar in the fermentation process, reducing the total sugar to 2.0%, and starting to add sugar, wherein the total sugar is controlled to be 1.5-2.5%.

And adding ammonia water according to the pH value, beginning to add ammonia water when the pH value is reduced to 6.0, and controlling the pH value to be 5.8-6.0.

Feeding soybean oil methyl ester oil after fermenting for 24hr, feeding at a feeding rate of 0.2g/hr/L, and stopping feeding 30hr before tank-placing, wherein the ratio of methyl ester oil to starch is 1: 12.

162hr, placing in a tank, with titer of 34820 μ/ml and fermentation index of 2.053, increasing fermentation level by 11.5%, reducing fermentation raw material cost by 15.5%, and removing residue of methyl esterification oil.

Example 2

The seed culture mode is not changed, and the fermentation culture condition is not changed.

The fermentation medium and the feed supplement select cottonseed oil methyl ester oil, and the methyl ester rate is 84.5 percent.

The fermentation medium comprises the following components: 4% of starch, 0.4% of methyl esterified grease, 2% of soybean cake powder, 2.2% of corn steep liquor, 0.45% of yeast powder, 0.75% of ammonium sulfate, 0.55% of light calcium carbonate and a proper amount of trace elements and inorganic salt, wherein the ratio of methyl esterified oil to starch in the base material is 1: 10.

And (3) adding starch sugar in the fermentation process, reducing the total sugar to 2.0%, and starting to add sugar, wherein the total sugar is controlled to be 1.5-2.5%.

And adding ammonia water according to the pH value, beginning to add ammonia water when the pH value is reduced to 6.0, and controlling the pH value to be 5.8-6.0.

And (3) after fermenting for 25hr, feeding cottonseed oil methyl ester oil at a feeding rate of 0.15g/hr/L, and stopping feeding 28hr before putting into a tank, wherein the ratio of the methyl ester oil to the starch in the whole process is 1: 10.

165hr, fermenting at 34890 μ/ml with fermentation index of 2.125, 12.2% higher fermentation level, 14.3% lower cost of raw materials, and no residue in methylated oil.

Example 3

The seed culture mode is not changed, and the fermentation culture condition is not changed.

The rapeseed oil methyl esterification oil is selected by the fermentation medium and the supplementary material, and the methyl ester rate is 86.7 percent.

The fermentation medium comprises the following components: 3% of starch, 0.35% of methyl esterified oil, 2.5% of soybean cake powder, 2.7% of corn steep liquor, 0.3% of yeast powder, 0.58% of ammonium sulfate, 0.6% of light calcium carbonate and a proper amount of trace elements and inorganic salts, wherein the ratio of the methyl esterified oil to the starch in the base material is 1: 11.6.

And (3) adding starch sugar in the fermentation process, reducing the total sugar to 2.0%, and starting to add sugar, wherein the total sugar is controlled to be 1.5-2.5%.

And adding ammonia water according to the pH value, beginning to add ammonia water when the pH value is reduced to 6.0, and controlling the pH value to be 5.8-6.0.

Feeding rapeseed oil methyl esterification oil after fermenting for 30hr, feeding at a feeding rate of 0.17g/hr/L, stopping feeding 25hr before putting into a tank, and feeding methyl ester oil and starch at a ratio of 1:11.

160hr, the mixture is put into a tank, the tank placing titer is 34290 mu/ml, the fermentation index is 2.103, the tank placing fermentation level is improved by 11.2 percent, the cost of fermentation raw materials is reduced by 12.5 percent, and the tank placing methyl esterification oil has no residue.

The above description is only a preferred embodiment of the present invention, and the present invention can provide a concept that different methyl esterified oils of vegetable oil can be applied to tetracycline fermentation medium and supplement under the premise of the present concept, and the methyl esterified oils obtained from different methyl ester ratios or different oils can be applied to tetracycline fermentation process, which should be regarded as the protection scope of the present invention.