阅读说明：本技术 一种申嗪霉素发酵工艺 (Shenqinmycin fermentation process ) 是由 张进武 郝祥蕊 冯镇泰 张红艳 龚新进 于 2020-07-01 设计创作，主要内容包括：本发明涉及微生物发酵领域,特别是涉及一种申嗪霉素发酵工艺,所述发酵工艺包括：向M18菌体发酵体系中补加碳源和/或氮源。所述碳源选自糖类、油脂、有机酸、有机酸酯或小分子醇。所述氮源选自无机氮源或有机氮源。所述无机氮源选自铵盐,所述铵盐优选为硫酸铵或硝酸铵,所述有机氮源选自复合氨基酸粉或酰胺。本发明的申嗪霉素发酵工艺维持菌体的连续生长和产物形成的需要,延缓菌体的衰老,将申嗪霉素大罐发酵周期延长至65小时左右；延长发酵产物PCA的合成与分泌周期,在延长期发酵产物PCA的含量仍继续大幅增长,最高含量可达9.7g/L以上；大大提高了发酵产量,降低了生产成本。(The invention relates to the field of microbial fermentation, in particular to a shenqinmycin fermentation process, which comprises the following steps: and (3) supplementing a carbon source and/or a nitrogen source into the M18 thallus fermentation system. The carbon source is selected from saccharides, grease, organic acid ester or small molecular alcohol. The nitrogen source is selected from inorganic nitrogen sources or organic nitrogen sources. The inorganic nitrogen source is selected from ammonium salt, the ammonium salt is preferably ammonium sulfate or ammonium nitrate, and the organic nitrogen source is selected from compound amino acid powder or amide. The fermentation process of the shenqinmycin maintains the continuous growth of thalli and the requirement of product formation, delays the aging of the thalli, and prolongs the fermentation period of the shenqinmycin in a large tank to about 65 hours; the synthesis and secretion period of the fermentation product PCA is prolonged, the content of the fermentation product PCA is still increased greatly in the prolonged period, and the highest content can reach more than 9.7 g/L; greatly improves the fermentation yield and reduces the production cost.)

1. A shenqinmycin fermentation process is characterized by comprising the following steps: and (3) supplementing a carbon source and/or a nitrogen source into the M18 thallus fermentation system.

2. The fermentation process of claim 1, wherein the M18 fermentation system is obtained by any method selected from the group consisting of:

1) inoculating M18 thallus seed bacterial liquid into a fermentation shake flask to obtain the microbial strain;

2) inoculating M18 thallus seed bacterial liquid into a seeding tank for culture, and transferring the thallus to a large tank for fermentation when the thallus concentration OD600 reaches more than 2.0.

3. The fermentation process of claim 1, wherein the carbon source is selected from the group consisting of sugars, lipids, organic acids and organic acid esters.

4. The fermentation process according to claim 3, wherein the saccharide is selected from the group consisting of monosaccharides, preferably glucose, or polysaccharides, preferably sucrose.

5. The fermentation process of claim 1, wherein the nitrogen source is selected from an inorganic nitrogen source or an organic nitrogen source.

6. Fermentation process according to claim 5, characterized in that the inorganic nitrogen source is chosen from ammonium salts, preferably ammonium sulfate or ammonium nitrate, and the organic nitrogen source is chosen from complex amino acid powders or amides.

7. The fermentation process of claim 1, wherein the additional carbon or nitrogen source is an aqueous solution of each.

8. The fermentation process according to claim 1, wherein when the content of reducing sugar or the content of amino nitrogen in the fermentation system is 70-75% of the initial mass concentration, the carbon source or the nitrogen source is added.

9. The fermentation process according to claim 1, wherein the total mass fraction of the supplemented carbon source is 0.2-1.0% and/or the total mass fraction of the supplemented nitrogen source is 0.2-0.8%, measured on the basis of the volume of the initial medium.

10. The fermentation process of claim 1, wherein the carbon or nitrogen source is fed in a manner selected from the group consisting of a fractionated feeding and a continuous feeding.

Technical Field

The invention relates to the field of microbial fermentation, in particular to a shenqinmycin fermentation process.

Background

The shenqinmycin is a phenazine agricultural broad spectrum bactericide, the chemical name of which is phenazine-1-carboxylic acid (PCA), and is obtained by utilizing growth-promoting antagonistic bacterium M18 to be cultured in a submerged layer in a fermentation tank and then extracted and purified. The Pseudomonas fluorescens (Pseudomonas fluorescens) of the fungus is preserved in China general microbiological culture Collection center, and the preservation number is as follows: CGMCC NO.0462, whose disclosure is incorporated herein by reference, describes a biopesticide growth-promoting antagonist M18 and a method for preparing the same in Chinese patent CN00119857.2, Qiyuquan, Von Zhentai, inventor.

At present, in the whole fermentation process of industrially producing PCA by using M18 bacteria, about 25 hours before fermentation is a bacteria growth and propagation stage, about 75% of nutrient components C (reducing sugar) and N (amino nitrogen) are consumed, then the fermentation stage enters a product PCA biosynthesis stage, after about 40 hours of fermentation, the nutrient components C are basically consumed, the bacteria growth is influenced, the bacteria growth starts to age and autolyze, the intracellular amino nitrogen and product resolvase are released to cause the content of the product PCA to start to decrease, the fermentation is finished, and the content of the product PCA in fermentation liquor is about 4.5 g/L.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a fermentation process of shenqinmycin, which solves the problems of the prior art.

In order to achieve the above objects and other related objects, the present invention provides a shenqinmycin fermentation process, which comprises: and (3) supplementing a carbon source and/or a nitrogen source into the M18 thallus fermentation system.

Preferably, the M18 fermentation system is obtained by a method selected from the group consisting of:

1) inoculating M18 thallus seed bacterial liquid into a fermentation shake flask to obtain the microbial strain;

2) inoculating M18 thallus seed bacterial liquid into a seeding tank for culture, and transferring the thallus to a large tank for fermentation when the thallus concentration OD600 reaches more than 2.0.

Preferably, the carbon source is selected from sugars, fats and oils, organic acids or organic acid esters.

Preferably, the saccharide is selected from a monosaccharide, preferably glucose, or a polysaccharide, preferably sucrose.

Preferably, the nitrogen source is selected from inorganic nitrogen sources or organic nitrogen sources.

Preferably, the inorganic nitrogen source is selected from ammonium salts, preferably ammonium sulfate or ammonium nitrate, and the organic nitrogen source is selected from complex amino acid powder or amide.

Preferably, the additional carbon or nitrogen source is a respective aqueous solution.

Preferably, when the content of reducing sugar or the content of amino nitrogen in the fermentation system is 70-75% of the initial mass concentration, the carbon source or the nitrogen source is added.

Preferably, the total amount of the supplemented carbon source is 0.2-1.0% and/or the total amount of the supplemented nitrogen source is 0.2-0.8% measured by taking the volume of the culture medium in the fermentation container as a base number.

Preferably, the carbon and/or nitrogen source is fed in a manner selected from a batchwise feeding or a continuous feeding.

As mentioned above, the fermentation process of shenqinmycin of the invention has the following beneficial effects:

1) the continuous growth of the thalli and the requirement of product formation are maintained, the aging of the thalli is delayed, and the fermentation period of the shenqinmycin in a large tank is prolonged to about 65 hours;

2) the synthesis and secretion period of the fermentation product PCA is prolonged, the content of the fermentation product PCA is still increased greatly in the prolonged period, and the highest content can reach more than 9.7 g/L;

3) greatly improves the fermentation yield and reduces the production cost.

Drawings

FIG. 1 is a graph showing the change of the contents of reducing sugar (C) and amino nitrogen (N) in the test of simultaneously supplementing carbon and nitrogen sources in portions during the cultivation in a fermenter.

FIG. 2 is a graph showing the change in PCA content in the experiment of simultaneous supply of carbon and nitrogen sources in divided portions during the cultivation in a fermenter.

FIG. 3 is a graph showing the change of the reducing sugar (C) and amino nitrogen (N) contents in the fermentor culture with continuous addition of carbon and nitrogen sources.

FIG. 4 is a graph showing the change in PCA content in the experiment in which the fermenter was cultured while continuously supplying the carbon source and the nitrogen source.

Detailed Description

The invention provides a shenqinmycin fermentation process, which comprises the following steps: and (3) supplementing a carbon source and/or a nitrogen source into the M18 thallus fermentation system.

The fermentation process further comprises one or several of the following conditions:

1) the culture medium used in the M18 fermentation system is a culture medium containing glucose and peptone;

2) the culture temperature is 26-30 ℃;

3) the rotation speed is 80-200 rpm.

The M18 fermentation system is obtained by any mode selected from the following modes:

1) inoculating M18 thallus seed bacterial liquid into a fermentation shake flask to obtain the microbial strain;

2) inoculating M18 thallus seed bacterial liquid into a seeding tank for culture, and transferring to a large tank for fermentation when the thallus concentration OD600 reaches more than 2.0.

The method for obtaining the seed bacterial liquid comprises the following steps: picking up a little of lawn from the slant of M18 strain, transferring to the slant of eggplant bottle, culturing at 28-30 deg.C for 24 hr, washing the lawn on the slant of eggplant bottle with sterilized distilled water, and sterilizing in triangular flask to obtain seed bacterial liquid.

The culture medium components of the M18 strain slant and the eggplant bottle slant are as follows: 10g/L of glucose, 5g/L of yeast extract powder, 10g/L of bone peptone, 1g/L of sodium chloride and 18g/L of agar. The components are prepared by pure water, the pH value is 7.0-7.2, the sterilization is carried out for 20min at the temperature of 115 ℃, and the components are placed and cooled to form an inclined plane.

Specifically, the shake flask culture medium in the mode 1) consists of: 5g/L of glycerol, 20g/L of glucose, 15g/L of bone peptone, 0.2g/L of dipotassium hydrogen phosphate and 1g/L of magnesium sulfate. The components are prepared by pure water, the pH value is 7.0-7.2, and the components are sterilized for 20min at the temperature of 115 ℃.

Specifically, the culture conditions of the fermentation shake flask in the mode 1) are as follows: the temperature of the shaking table is 28-30 ℃, and the rotating speed is 200 r/min.

Specifically, the composition of the seeding tank culture medium in the mode 2) is as follows: 10g/L of glucose, 5g/L of yeast extract powder, 10g/L of bone peptone, 1g/L of sodium chloride and the balance of water, wherein the pH value is 7.0-7.2, and the sterilization is carried out for 20min at the temperature of 115 ℃.

Specifically, the culture temperature of the seeding tank in the mode 2) is 28-30 ℃.

The pressure of the seed tank is 0.02-0.03 MPa.

The air flow of the seeding tank is 1: 0.1-1: 1.5 (the ratio of the volume of the seeding tank to the volume of air per minute).

The stirring speed of the seeding tank is 100-150 rpm.

Generally, after culturing for 22-24 hours, sampling and microscopic examination are carried out, the thallus grows vigorously, no foreign bacteria pollution is caused, and the thallus concentration OD600 can reach more than 2.0.

Specifically, when the seeds are transferred to a large tank for fermentation in the mode 2), the inoculation amount is 1-2% of the volume of the large tank culture medium.

Specifically, the composition of the large-tank culture medium is as follows: 5g/L of glycerol, 20g/L of glucose, 15g/L of bone peptone, 0.2g/L of dipotassium hydrogen phosphate, 1g/L of magnesium sulfate and the balance of water, wherein the pH value is 6.5-7.5, and the sterilization is carried out for 20min at the temperature of 115 ℃.

Specifically, the culture temperature of the large tank is 28-30 ℃.

The tank pressure of the large tank is 0.01-0.03 MPa.

The air flow rate of the large tank is 1: 0.1-1: 1.5 (the ratio of the large tank volume to the air volume per minute).

The stirring speed of the large tank is 80-120 rpm.

The M18 belongs to Pseudomonas fluorescens (Pseudomonas fluorescens), is the same as M18 in Chinese patent CN00119857.2, and is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.0462 when the patent CN00119857.2 is applied.

The carbon source is a nutrient substance which contains carbon element and can be utilized by the growth and the reproduction of microorganisms, and the carbon source comprises sugar, grease, organic acid or organic acid ester. Wherein the saccharide comprises monosaccharide and polysaccharide, the monosaccharide can be glucose, and the polysaccharide can be sucrose; the organic acid ester is an ester formed by reacting an organic acid with an alcohol.

The nitrogen source is a material for synthesizing substances such as amino acids, proteins, nucleic acids, nitrogen-containing metabolites and the like of the somatic cells. Specifically, the nitrogen source includes inorganic nitrogen sources and organic nitrogen sources. The inorganic nitrogen source comprises ammonium salt, the ammonium salt can be ammonium sulfate and ammonium nitrate, and the organic nitrogen source can be compound amino acid powder and amide.

The solvent of the carbon source and the nitrogen source is water, and the carbon source and the nitrogen source are used after sterilization.

And when the content of reducing sugar or the content of amino nitrogen in the fermentation system is 70-75% of the initial mass concentration, beginning to supplement a carbon source or a nitrogen source. The content of the reducing sugar is determined by a film reagent method, and the content of the amino nitrogen is determined by a formaldehyde titration method.

During the fermentation culture in a large tank, sampling every 2 hours to detect the contents of reducing sugar, amino nitrogen and a product shenqinmycin (PCA). Generally, the mixture is transferred into a large tank for fermentation for about 25 hours, reducing sugar and amino nitrogen consume 70-75% of the initial mass concentration, and then carbon sources or nitrogen sources can be supplemented.

The total mass fraction of the supplemented carbon source is 0.2-1.0%, for example 0.6%, measured by taking the volume of the initial culture medium as a base, and the unit is kg/L or g/mL, and the total mass of the supplemented carbon source is the mass of the powder before dissolution with water.

The total amount of the supplemented nitrogen source is 0.2-0.8%, such as 0.5%, measured by taking the volume of the initial culture medium as a base, and the unit is kg/L or g/mL, and the total mass of the supplemented nitrogen source is the mass of the powder before dissolution with water.

The carbon source or the nitrogen source can be supplemented for several times, for example, for two times, wherein each time, the total amount of the carbon source or the nitrogen source is half of the total amount; or adding the powder three times, wherein 1/3 in the total amount is added each time. The supplemented carbon source or nitrogen source can also be continuously supplemented (or called as continuous supplement), the flow rate of the supplemented materials is controlled, and the supplementation is completed within a certain time before the fermentation is finished. When the carbon source and the nitrogen source are supplemented, the carbon source and the nitrogen source may be added after being mixed or may be added separately.

After 0.6 percent of carbon source and 0.5 percent of nitrogen source are supplemented in a divided or continuous way, the yield of PCA reaches over 9.7g/L when the fermentation is carried out for 65 hours.

The yield of PCA was measured by HPLC and the detection method was as follows:

taking 200 mul of zymophyte liquid, adding a proper amount of acetonitrile to dilute the zymophyte liquid in a 10ml volumetric flask, oscillating the zymophyte liquid in an ultrasonic bath for 15min, recovering to room temperature, fixing the volume and shaking up the zymophyte liquid. Taking 1ml of sample to a graduated centrifuge tube, centrifuging for 10min at 15000 r/min, taking 10 μ l of supernatant, and carrying out sample injection detection under the detection condition of 248nm wavelength and about 14.5min retention time; the mobile phase was acetonitrile, water, glacial acetic acid 40:60:0.2, flow rate 1.0ml/min, C18 reverse column.

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

It should be understood that the processing equipment or devices not specifically mentioned in the following examples are conventional in the art; all pressure values and ranges refer to absolute pressures.

Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.