CN105506040A - Method for fermentation production of enramycin - Google Patents
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Abstract
The invention relates to a method for fermentation production of enramycin. During the fermentation process of constant temperature culture in a fermentation culture medium, an inorganic salt solution is added in batches at different stages, synthesis of enramycin is promoted, oxygen dissolution of fermentation liquor can be improved, and the fermentation level of enramycin can be increased. The method is simple and feasible, and after fermentation of the process in the fermentation culture process, the fermentation level of enramycin can be increased by 25%.
Description
Technical field
The present invention relates to fermentation technical field, be specially a kind of method of producing enramycin by fermentation.
Background technology
Enramycin (enramycin) have another name called enramycin or and enramycin, be the polypeptide class poultry microbiotic produced by kabicidin streptomycete fermentation.Enramycin is by the organic bases comprising 13 different types of 17 amino acid moleculars and fatty acid molecule and form.Main ingredient has enramycin A and B, is the application of its hydrochloride form.Enramycin has very high stability in feed, be not degraded in enteron aisle, to the effect of gram-positive microorganism and strong, life-time service can not produce resistance, and it can change the community distribution of bacterium in enteron aisle, the dietetic alimentation of favourable forage component, can promote the weight of animals to increase and improve the utilization ratio of feed.Enramycin was researched and developed in 1996 by Japan's military field pharmaceutical industries strain formula, was registered and widely used thereafter in many countries.2005, domestic production enterprise and offshore company started joint production enramycin premix.Along with other are eliminated by microbiotic step, the use of enramycin will certainly be more and more extensive, but the industrial production of enramycin faces the low problem of capacity and output always.
Summary of the invention
The object of the present invention is to provide a kind of method of producing enramycin by fermentation, for overcoming the defect existed in prior art.
For achieving the above object, the invention provides a kind of method of producing enramycin by fermentation, by in the fermenting process of constant temperature culture in the fermentation medium, different steps portion-wise addition inorganic salt solution, promote that the synthesis of enramycin also can improve the dissolved oxygen of fermented liquid, improve the fermentation level of enramycin.
Preferably, described leavening temperature is 28 DEG C, and rotating speed is 250 ~ 350r/min.
Preferably, described inorganic salt are magnesium sulfate and/or ammonium chloride.
Preferably, the addition of described inorganic salt solution is the 1.5%-5% of fermentating liquid volume.
Preferably, described inorganic salt solution be containing magnesium sulfate and ammonium chloride mixed solution; Wherein magnesium sulfate concentration is 0.05-0.1g/L, and ammonium chloride concentration is 0.1-0.3g/L.
Preferably, the time phase adding described inorganic salt solution be fermentation 52 hours, 64 hours, 72 hours.
Preferably, in fermenting process, constantly little in fermentation 52, the concentration of adding magnesium sulfate is 0.05-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.1-0.3g/L and the volume of mixed solution is the 1.5%-5% of fermentating liquid volume; Fermentation 64 hours, the concentration of adding magnesium sulfate was 0.05-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.1-0.3g/L and the volume of mixed solution is the 1.5%-5% of fermentating liquid volume; Constantly little in fermentation 72, the concentration of adding magnesium sulfate is 0.05-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.1-0.3g/L and the volume of mixed solution is the 1.5%-5% of fermentating liquid volume; Thus the synthesis of enramycin can be beneficial to better and control dissolved oxygen in fermented liquid, improve fermentation production strength, improve and accelerate the production of enramycin.
Further preferably, in fermenting process, constantly little in fermentation 52, the concentration of adding magnesium sulfate is 0.08-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.2-0.3g/L and the volume of mixed solution is the 2%-5% of fermentating liquid volume; Constantly little in fermentation 64, the concentration of adding magnesium sulfate is 0.08-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.2-0.3g/L and the volume of mixed solution is the 2%-5% of fermentating liquid volume; Constantly little in fermentation 72, the concentration of adding magnesium sulfate is 0.08-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.2-0.3g/L and the volume of mixed solution is the 2%-5% of fermentating liquid volume.
More preferably, in fermenting process, constantly little in fermentation 52, the concentration of adding magnesium sulfate is 0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.3g/L and the volume of mixed solution is 3% of fermentating liquid volume; Constantly little in fermentation 64, the concentration of adding magnesium sulfate is 0.05g/L, and the concentration of ammonium chloride is the mixed solution of 0.3g/L and the volume of mixed solution is 3% of fermentating liquid volume; Constantly little in fermentation 72, the concentration of adding magnesium sulfate is 0.05g/L, and the concentration of ammonium chloride is the mixed solution of 0.3g/L and the volume of mixed solution is 3% of fermentating liquid volume.
The bacterial strain of the enramycin for fermentative production that the present invention relates to is kabicidin streptomycete (Streptomycesfungicidious) FYFJ03, and preserving number is CGMCCNo.4113, open in Chinese patent CN101974469A.
In the method for producing enramycin by fermentation of the present invention, inoculation is first cultivated in 25-30 DEG C of constant temperature culture by the bacterial strain enlarged culturing stage on solid slant culture base; With sterilized water bacterium washed down again and make in spore suspension inoculation fermentation substratum.Wherein, described solid medium comprises following component: maltose 10g/L, dried yeast powder 1g/L, and tuna cream 1g/L, Tryptones 1g/L, agar is appropriate.
In fermentation culture process of the present invention, the fermention medium of described producing enramycin by fermentation comprises following component: Semen Maydis powder 80-120g/L, corn steep liquor 8-12g/L, ammonium sulfate 2.5-3.5g/L, zinc sulfate 0.05-0.15g/L, calcium carbonate 18-22g/L.
Preferably, the fermention medium of described producing enramycin by fermentation comprises following component: Semen Maydis powder 100g/L, corn steep liquor 10g/L, ammonium sulfate 3g/L, zinc sulfate 0.1g/L, calcium carbonate 20g/L.
Research finds, adds the synthesis that inorganic salt can promote enramycin during the fermentation, improves fermentation level; In addition, produce the bacterial strain of enramycin very high to the demand of oxygen, when the too high meeting of cell concentration causes fermenting process dissolved oxygen under-supply, affect output, bacteria concentration is too low, and enramycin output is low, and fermentation period is long, and the aging and easily microbiological contamination of thalline, affects production.So not only the mixed solution of one or more inorganic salt of portion-wise addition is beneficial to the synthesis of enramycin but also is beneficial to the dissolved oxygen amount improving fermented liquid during the fermentation, improve fermentation level.
Not only the method for producing enramycin by fermentation provided by the invention is beneficial to the synthesis of enramycin in fermenting process but also solves the problem of dissolved oxygen amount deficiency in strain fermentation process, operation is simple, by after the fermentation of this technique in fermentation culture process, the fermentation level that the method enramycin of inorganic salt is added in more non-feed supplement can improve about 25%, effectively improves fermentation level.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
If do not specialize, technique means used in the embodiment conventional means known by those skilled in the art.The each raw material added unless otherwise indicated, is commercially available.
Embodiment 1
1) enlarged culturing of bacterial strain:
The frozen bacterium liquid getting bacterial strain FYFJ03 is diluted to 10
6individual spore/ml spore liquid, coats Solid media for plates, cultivates 7 days for 28 DEG C, chooses bacterium shape rule, well-grown single strain is inoculated on solid slant culture base, cultivates 8 days for 28 DEG C.
Wherein, described solid medium comprises following component: maltose 10g/L, dried yeast powder 1g/L, tuna cream 1g/L, Tryptones 1g/L, agar 18g/L.
2) fermentation culture:
By step 1) bacterial classification cultivated aseptically is inoculated in the 50L ferment tank substratum of first sterilizing, 28 DEG C of cultivations, fermentation period 8 days.The concentration that magnesium sulfate is added in beginning in 52 hours batch of fermenting is 0.05g/L, and the concentration of ammonium chloride is the mixed solution of 0.1g/L and the volume of mixed solution is 1.5% of fermentating liquid volume; The concentration that magnesium sulfate is added in beginning in 64 hours batch of fermenting is 0.05g/L, and the concentration of ammonium chloride is the mixed solution of 0.1g/L and the volume of mixed solution is 1.5% of fermentating liquid volume; The concentration that magnesium sulfate is added in beginning in 72 hours batch of fermenting is 0.05g/L, and the concentration of ammonium chloride is the mixed solution of 0.1g/L and the volume of mixed solution is 1.5% of fermentating liquid volume.Ferment the horizontal 6100U/ml of average fermentation after 8 days
Wherein, described fermention medium comprises following component: Semen Maydis powder 100g/L, corn steep liquor 10g/L, ammonium sulfate 3g/L, zinc sulfate 0.1g/L, calcium carbonate 20g/L.
Embodiment 2
The frozen bacterium liquid getting bacteria strain FYFJ03 is diluted to 10
6individual spore/ml spore liquid, coats Solid media for plates, cultivates 7 days for 28 DEG C, chooses bacterium shape rule, well-grown single strain is inoculated on solid slant culture base, cultivates 7 days for 28 DEG C.
Wherein, described solid medium comprises following component: Semen Maydis powder 20g/L, peptone 1g/L, zinc sulfate 0.1g/L, Manganous chloride tetrahydrate 0.02g/L, agar 15g/L.
2) fermentation culture:
By step 1) bacterial classification cultivated aseptically is inoculated in the 50L ferment tank substratum of first sterilizing, 28 DEG C of cultivations, fermentation period 8 days.The concentration that magnesium sulfate is added in beginning in 52 hours batch of fermenting is 0.08g/L, and the concentration of ammonium chloride is the mixed solution of 0.2g/L and the volume of mixed solution is 2% of fermentating liquid volume; The concentration that magnesium sulfate is added in beginning in 64 hours batch of fermenting is 0.08g/L, and the concentration of ammonium chloride is the mixed solution of 0.2g/L and the volume of mixed solution is 2% of fermentating liquid volume; The concentration that magnesium sulfate is added in beginning in 72 hours batch of fermenting is 0.08g/L, and the concentration of ammonium chloride is the mixed solution of 0.2g/L and the volume of mixed solution is 2% of fermentating liquid volume.Ferment the horizontal 6640U/ml of average fermentation after 8 days
Wherein, described fermention medium comprises following component: Semen Maydis powder 100g/L, corn steep liquor 10g/L, ammonium sulfate 3g/L, zinc sulfate 0.1g/L, calcium carbonate 20g/L.
Embodiment 3
The frozen bacterium liquid getting bacterial strain FYFJ03 is diluted to 10
5individual spore/ml spore liquid, coats Solid media for plates, cultivates 7 days for 28 DEG C, chooses bacterium shape rule, well-grown single strain is inoculated on solid slant culture base, cultivates 7 days for 28 DEG C.
Wherein, described solid medium comprises following component: Semen Maydis powder 20g/L, peptone 1g/L, zinc sulfate 0.1g/L, Manganous chloride tetrahydrate 0.02g/L, agar 15g/L.
2) fermentation culture:
By step 1) bacterial classification cultivated aseptically is inoculated in the 50L ferment tank substratum of first sterilizing, 28 DEG C of cultivations, fermentation period 8 days.The concentration that magnesium sulfate is added in beginning in 52 hours batch of fermenting is 0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.3g/L and the volume of mixed solution is 3% of fermentating liquid volume; The concentration that magnesium sulfate is added in beginning in 64 hours batch of fermenting is 0.05g/L, and the concentration of ammonium chloride is the mixed solution of 0.3g/L and the volume of mixed solution is fermentating liquid volume 3%; The concentration that magnesium sulfate is added in beginning in 72 hours batch of fermenting is 0.05g/L, and the concentration of ammonium chloride is the mixed solution of 0.3g/L and the volume of mixed solution is 3% of fermentating liquid volume.Ferment the horizontal 7190U/ml of average fermentation after 8 days
Wherein, described fermention medium comprises following component: Semen Maydis powder 100g/L, corn steep liquor 10g/L, ammonium sulfate 3g/L, zinc sulfate 0.1g/L, calcium carbonate 20g/L.
Comparative example
1) slant culture:
The frozen bacterium liquid getting bacterial strain FYFJ03 is diluted to 10
5individual spore/ml spore liquid, coats Solid media for plates, cultivates 7 days for 28 DEG C, chooses bacterium shape rule, well-grown single strain is inoculated on solid slant culture base, cultivates 7 days for 28 DEG C.
Wherein, described solid medium comprises following component: Semen Maydis powder 20g/L, peptone 1g/L, zinc sulfate 0.1g/L, Manganous chloride tetrahydrate 0.02g/L, agar 15g/L.
2) fermentation culture:
By step 1) be inoculated on the fermention medium of 50L tank under the bacterial classification aseptic condition cultivated, 28 DEG C, 300r/min, in fermentation culture process, does not add the mixed solution of magnesium sulfate and ammonium chloride, and fermentation number of days is 8 days, the horizontal 5750U/ml of average fermentation.
Wherein, described fermention medium comprises following component: Semen Maydis powder 100g/L, corn steep liquor 10g/L, ammonium sulfate 3g/L, zinc sulfate 0.1g/L, calcium carbonate 20g/L.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a method for producing enramycin by fermentation, is characterized in that, by the fermenting process of constant temperature culture in the fermentation medium, and different steps portion-wise addition inorganic salt solution.
2. method according to claim 1, is characterized in that, described leavening temperature is 28 DEG C, and rotating speed is 250 ~ 350r/min.
3. method according to claim 1 and 2, is characterized in that, described inorganic salt are magnesium sulfate and/or ammonium chloride; Preferably, the addition of described inorganic salt solution is the 1.5%-5% of fermentating liquid volume.
4. method according to claim 1 and 2, is characterized in that, described inorganic salt solution be containing magnesium sulfate and ammonium chloride mixed solution; Wherein magnesium sulfate concentration is 0.05-0.1g/L, and ammonium chloride concentration is 0.1-0.3g/L.
5. method according to claim 1 and 2, is characterized in that, the time phase adding described inorganic salt solution be fermentation 52 hours, 64 hours, 72 hours.
6. method according to claim 1 and 2, it is characterized in that, in fermenting process, constantly little in fermentation 52, the concentration of adding magnesium sulfate is 0.05-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.1-0.3g/L and the volume of this mixed solution is the 1.5%-5% of fermentating liquid volume; Constantly little in fermentation 64, the concentration of adding magnesium sulfate is 0.05-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.1-0.3g/L and the volume of this mixed solution is the 1.5%-5% of fermentating liquid volume; Constantly little in fermentation 72, the concentration of adding magnesium sulfate is 0.05-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.1-0.3g/L and the volume of this mixed solution is the 1.5%-5% of fermentating liquid volume.
7. method according to claim 1 and 2, it is characterized in that, in fermenting process, constantly little in fermentation 52, the concentration of adding magnesium sulfate is 0.08-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.2-0.3g/L and the volume of this mixed solution is the 2%-5% of fermentating liquid volume; Constantly little in fermentation 64, the concentration of adding magnesium sulfate is 0.08-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.2-0.3g/L and the volume of this mixed solution is the 2%-5% of fermentating liquid volume; Constantly little in fermentation 72, the concentration of adding magnesium sulfate is 0.08-0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.2-0.3g/L and the volume of this mixed solution is the 2%-5% of fermentating liquid volume.
8. method according to claim 1 and 2, is characterized in that, in fermenting process, constantly little in fermentation 52, and the concentration of adding magnesium sulfate is 0.1g/L, and the concentration of ammonium chloride is the mixed solution of 0.3g/L and the volume of this mixed solution is 3% of fermentating liquid volume; Constantly little in fermentation 64, the concentration of adding magnesium sulfate is 0.05g/L, and the concentration of ammonium chloride is the mixed solution of 0.3g/L and the volume of this mixed solution is 3% of fermentating liquid volume; Constantly little in fermentation 72, the concentration of adding magnesium sulfate is 0.05g/L, and the concentration of ammonium chloride is the mixed solution of 0.3g/L and the volume of this mixed solution is 3% of fermentating liquid volume.
9. the method according to any one of claim 1-8, is characterized in that, the bacterial strain of the described enramycin for fermentative production is kabicidin streptomycete (Streptomycesfungicidious) FYFJ03, and preserving number is CGMCCNo.4113.
10. the method according to any one of claim 1-9, is characterized in that, inoculation is first cultivated in 25-30 DEG C of constant temperature culture by the bacterial strain enlarged culturing stage on solid slant culture base; Being washed down by bacterium with sterilized water makes in spore suspension inoculation fermentation substratum again; Wherein, described solid medium comprises following component: maltose 10g/L, dried yeast powder 1g/L, tuna cream 1g/L, Tryptones 1g/L, and agar is appropriate;
In fermentation culture process, the fermention medium of described producing enramycin by fermentation comprises following component: Semen Maydis powder 80-120g/L, corn steep liquor 8-12g/L, ammonium sulfate 2.5-3.5g/L, zinc sulfate 0.05-0.15g/L, calcium carbonate 18-22g/L;
Preferably, the fermention medium of described producing enramycin by fermentation comprises following component: Semen Maydis powder 100g/L, corn steep liquor 10g/L, ammonium sulfate 3g/L, zinc sulfate 0.1g/L, calcium carbonate 20g/L.
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