CN102943101B - Method for producing enramycin by fermenting - Google Patents
Method for producing enramycin by fermenting Download PDFInfo
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- CN102943101B CN102943101B CN201210466719.5A CN201210466719A CN102943101B CN 102943101 B CN102943101 B CN 102943101B CN 201210466719 A CN201210466719 A CN 201210466719A CN 102943101 B CN102943101 B CN 102943101B
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Abstract
The invention relates to a method for producing enramycin by fermenting. According to the method, bactericidin streptomycete FYFJ03 is taken as a fermenting strain; during a fermenting process for constant temperature culturing, different sugar concentrations are fed under different stages; the dissolved oxygen of fermentation liquor is improved; and the fermentation level of the enramycin is increased. The method is simple and easy to carry out. After the fermentation is performed according to the method during the fermenting culturing process, the fermentation level of the enramycin is increased by about 20%.
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, Enramycin, enramycin, enramycin, it belongs to a kind of polypeptide antibiotics, molecule is structure in the form of a ring, it is the polypeptide antibiotics that unsaturated fatty acids is combined with tens seed amino acids, amino acid molecular forms ring type polypeptide, on aspartic acid endways, connect fatty acid molecule, according to the fatty acid molecule difference of molecular end, can be divided into enramycin A and enramycin B, general said enramycin is the mixture of these two kinds of components, enramycin is a kind of polypeptide antibiotics by separated actinomycetes (Streptomyces fungiedious) fermentative production obtaining from soil.He has stronger restraining effect to gram-positive microorganism, and the mechanism of action is hinder gram-positive bacteria cell wall synthetic.Its sensitive bacterial mainly contains the various gram-positive coccis such as streptococcus aureus, Bacillus subtilus, charcoal maggot bacillus clostridium tetani, Clostridium botulinum, clostridium perfringens etc.Long-term a large amount of Enramycin that uses is difficult for developing immunity to drugs, and the bacterium colony that it can improve bacterium in animal intestinal distributes, be conducive to animal digesting and assimilating feed, can promote the increase of the weight of animals and the utilization ratio of feed, therefore microbiotic and the growth promoter that this medicine is livestock and poultry recommended by many countries.Another feature of enramycin is to be difficult for being absorbed by animal, seldom residual in livestock and poultry body.Enramycin was researched and developed by military field pharmaceutical industries Zhu Shi commercial firm in 1966, and in 1974 at Japanese official registration, many by national registration be widely used thereafter.China Ministry of Agriculture is in approval Gai Yao China in 1993 registration.2005, domestic production enterprise and U.S. Schering Plough animal health-care product company limited (Schering Plough Animal Health Corp.) started joint production enramycin premix.
Produce enramycin bacterial strain very high to the demand of oxygen, when the too high meeting of cell concentration causes fermenting process dissolved oxygen under-supply, affect fermentative production intensity.If bacteria concentration is too low, enramycin output is slower, and fermentation period is long, and thalline is aging, does not reach better effects.So the concentration that needs in producing to flow sugaring by changes is improved the dissolved oxygen amount of fermented liquid, raising fermentation level.
Summary of the invention
The object of the present invention is to provide a kind of method of producing enramycin by fermentation, to overcome the defect existing in above-mentioned prior art.
The bacterial strain for producing enramycin by fermentation the present invention relates to is kabicidin streptomycete (Streptomyces fungicidious) FYFJ03, and preserving number is CGMCC No.4113, open in Chinese patent CN101974469A.
The method of producing enramycin by fermentation provided by the invention is by making in the fermenting process of fermentation strain FYFJ03 constant temperature culture in fermention medium, different steps respectively stream add different sugared concentration, thereby can improve the dissolved oxygen in fermented liquid, realize the technique effect of the fermentation level that improves enramycin.
In the method for producing enramycin by fermentation of the present invention, fermenting process is: will ferment by inoculation on solid slant culture base, and 25-30 ℃ of constant temperature culture.
Wherein, the sugar that stream adds is amylum hydrolysate of the sugar.
Preferably, the sugar that stream adds is glucose, maltose, dextrin.
Wherein, in fermenting process, after fermentation 36～60 hours, stream adds the sugar that concentration is 200～300g/L; After fermentation 60～90 hours, stream adds the sugar that concentration is 350～450g/L; After fermentation 90 hours, stream adds the sugar that concentration is 500～600g/L, thereby dissolved oxygen in controlled fermentation liquid preferably improves fermentative production intensity, improves and accelerate the production of enramycin, reduces fermentation period, realizes good technique effect.
Preferably, in fermenting process, after fermentation 36-60 hour, stream adds the sugar that concentration is 225-275g/L; After fermentation 60-90 hour, stream adds the sugar that concentration is 375-425g/L; After fermentation 90 hours, stream adds the sugar that concentration is 525-575g/L.
More preferably, in fermenting process, after fermentation 36-60 hour, stream adds the sugar that concentration is 250g/L; After fermentation 60-90 hour, stream adds the sugar that concentration is 400g/L; After fermentation 90 hours, stream adds the sugar that concentration is 550g/L.
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.
The present invention is used for the fermention medium of producing enramycin by fermentation and 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.
The method of producing enramycin by fermentation provided by the invention has solved the problem of dissolved oxygen amount deficiency in strain fermentation process, operation is simple, after fermenting by this technique in fermentation culture process, compared with usual way (maintain stream sugaring concentration constant until fermentation) enramycin fermentation level improve approximately 20%, effectively improved fermentation level.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art, in embodiment 1-5 and comparative example, each raw material adding, except special instruction, is commercially available.In following examples, stream sugaring used is dextrin.
Embodiment 1
1) enlarged culturing of bacterial strain:
At 25 ℃ of room temperatures, get the slant pore of bacterial strain FYFJ03, be diluted to 10
7individual spore/ml spore suspension, coats solid medium, cultivates 7 days for 28 ℃, choose well-grown single strain and be inoculated in liquid fermentation medium, in 28 ℃, 220rpm shaking table is cultivated 160h, the output that detects enramycin in fermented liquid, fermentation unit is 4000U/ml.
2) slant culture:
By 1) single strain that filters out is inoculated on solid slant culture base under aseptic condition, 28 ℃ of constant temperature culture 8 days.
Wherein said solid slant culture base comprises following component: Semen Maydis powder 20g/L, peptone 1g/L, zinc sulfate 0.1g/L, g/L, Manganous chloride tetrahydrate 0.02g/L, agar 15g/L.
3) fermentation culture:
By 1) be inoculated in the 50L ferment tank substratum of sterilizing in advance 28 ℃ of cultivations under the bacterial classification aseptic condition cultivated.Fermentation 36h starts to flow sugaring, adjusts stage by stage sugared stream and adds concentration, fermentation period 8 days.Ferment 36～60 hours, stream adds the sugar that concentration is 250g/L; Ferment 60～90 hours, stream adds the sugar that concentration is 400g/L; After fermentation 90 hours, stream adds the sugar that concentration is 550g/L.The average fermentation level after 8 days of fermenting is 6240U/ml.
Wherein said 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
1) enlarged culturing of bacterial strain:
At room temperature, get the slant pore of bacterial strain FYFJ03, be diluted to 10
5individual spore/ml spore suspension, coats solid medium, cultivates 7 days for 28 ℃, choose well-grown single strain and be inoculated in liquid fermentation medium, in 28 ℃, 200rpm shaking table is cultivated 170h, the output that detects enramycin in fermented liquid, fermentation unit is 5000U/ml.
2) slant culture:
By 1) single strain that filters out is inoculated on solid slant culture base under aseptic condition, 25 ℃ of constant temperature culture 8 days.
Wherein said solid slant culture base comprises following component: Semen Maydis powder 20g/L, peptone 1g/L, zinc sulfate 0.1g/L, g/L, Manganous chloride tetrahydrate 0.02g/L, agar 15g/L.
3) fermentation culture:
By 1) be inoculated in the 100L ferment tank substratum of sterilizing in advance 25 ℃ of cultivations under the bacterial classification aseptic condition cultivated.Fermentation 36h starts to flow sugaring, adjusts stage by stage sugared stream and adds concentration, fermentation period 8 days.Ferment 36～60 hours, stream adds the sugar that concentration is 200g/L; Ferment 60～90 hours, stream adds the sugar that concentration is 350g/L; After fermentation 90 hours, stream adds the sugar that concentration is 500g/L.The average fermentation level after 8 days of fermenting is 5450U/ml.
Wherein said fermention medium comprises following component: Semen Maydis powder 80g/L, corn steep liquor 9g/L, ammonium sulfate 3.5g/L, zinc sulfate 0.15g/L, calcium carbonate 18g/L.
Embodiment 3
1) enlarged culturing of bacterial strain:
At room temperature, get the slant pore of bacterial strain FYFJ03, be diluted to 10
5individual spore/ml spore suspension, coats solid medium, cultivates 7 days for 29 ℃, choose well-grown single strain and be inoculated in liquid fermentation medium, in 29 ℃, 210rpm shaking table is cultivated 180h, the output that detects enramycin in fermented liquid, fermentation unit is 5000U/ml.
2) slant culture:
By 1) single strain that filters out is inoculated on solid slant culture base under aseptic condition, 30 ℃ of constant temperature culture 8 days.
Wherein said solid slant culture base comprises following component: Semen Maydis powder 20g/L, peptone 1g/L, zinc sulfate 0.1g/L, g/L, Manganous chloride tetrahydrate 0.02g/L, agar 15g/L.
3) fermentation culture:
By 1) be inoculated in the 50L ferment tank substratum of sterilizing in advance 30 ℃ of cultivations under the bacterial classification aseptic condition cultivated.Fermentation 36h starts to flow sugaring, adjusts stage by stage sugared stream and adds concentration, fermentation period 8 days.Ferment 36～60 hours, stream adds the sugar that concentration is 300g/L; Ferment 60～90 hours, stream adds the sugar that concentration is 450g/L; After fermentation 90 hours, stream adds the sugar that concentration is 600g/L.The average fermentation level after 8 days of fermenting is 5860U/ml.
Wherein said fermention medium comprises following component: Semen Maydis powder 120g/L, corn steep liquor 12g/L, ammonium sulfate 2.8g/L, zinc sulfate 0.12g/L, calcium carbonate 22g/L.
Embodiment 4
1) enlarged culturing of bacterial strain:
At room temperature, get the slant pore of bacterial strain FYFJ03, be diluted to 10
6individual spore/ml spore suspension, coats solid medium, cultivates 7 days for 28 ℃, choose well-grown single strain and be inoculated in liquid fermentation medium, in 28 ℃, 215rpm shaking table is cultivated 165h, the output that detects enramycin in fermented liquid, fermentation unit is 4900U/ml.
2) slant culture:
By 1) single strain that filters out is inoculated on solid slant culture base under aseptic condition, 28 ℃ of constant temperature culture 8 days.
Wherein said solid slant culture base comprises following component: Semen Maydis powder 20g/L, peptone 1g/L, zinc sulfate 0.1g/L, g/L, Manganous chloride tetrahydrate 0.02g/L, agar 15g/L.
3) fermentation culture:
By 1) be inoculated in the 50L ferment tank substratum of sterilizing in advance 27 ℃ of cultivations under the bacterial classification aseptic condition cultivated.Fermentation 36h starts to flow sugaring, adjusts stage by stage sugared stream and adds concentration, fermentation period 8 days.Ferment 36～60 hours, stream adds the sugar that concentration is 275g/L; Ferment 60～90 hours, stream adds the sugar that concentration is 425g/L; After fermentation 90 hours, stream adds the sugar that concentration is 550g/L.The average fermentation level after 8 days of fermenting is 6040U/ml.
Wherein said fermention medium comprises following component: Semen Maydis powder 90g/L, corn steep liquor 11g/L, ammonium sulfate 3.2g/L, zinc sulfate 0.05g/L, calcium carbonate 19g/L.
Embodiment 5
1) enlarged culturing of bacterial strain:
At room temperature, get the slant pore of bacterial strain FYFJ03, be diluted to 10
6individual spore/ml spore suspension, coats solid medium, cultivates 7 days for 28 ℃, choose well-grown single strain and be inoculated in liquid fermentation medium, in 28 ℃, 200rpm shaking table is cultivated 175h, the output that detects enramycin in fermented liquid, fermentation unit is 5100U/ml.
2) slant culture:
By 1) single strain that filters out is inoculated on solid slant culture base under aseptic condition, 28 ℃ of constant temperature culture 8 days.
Wherein said solid slant culture base comprises following component: Semen Maydis powder 20g/L, peptone 1g/L, zinc sulfate 0.1g/L, g/L, Manganous chloride tetrahydrate 0.02g/L, agar 15g/L.
3) fermentation culture:
By 1) be inoculated in the 50L ferment tank substratum of sterilizing in advance 29 ℃ of cultivations under the bacterial classification aseptic condition cultivated.Fermentation 36h starts to flow sugaring, adjusts stage by stage sugared stream and adds concentration, fermentation period 8 days.Ferment 36～60 hours, stream adds the sugar that concentration is 250g/L; Ferment 60～90 hours, stream adds the sugar that concentration is 400g/L; After fermentation 90 hours, stream adds the sugar that concentration is 550g/L.The average fermentation level after 8 days of fermenting is 6100U/ml.
Wherein said fermention medium comprises following component: Semen Maydis powder 100g/L, corn steep liquor 9g/L, ammonium sulfate 2.5g/L, zinc sulfate 0.15g/L, calcium carbonate 18g/L.
Comparative example
Strain expanded culture and slant culture are referring to the corresponding method of embodiment 1～5, and different is in fermentation culture process, keep stream sugaring concentration constant.Fermentation starts to add the sugar of 500g/L for 36 hours, and maintaining stream, to add concentration constant, until fermentation ends.The average fermentation level after 8 days of fermenting is 5210U/ml.
Above embodiment is used for illustrating the present invention, but is not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, modification made for the present invention or replacement, all belong to scope of the present invention.
Claims (5)
1. the method for a producing enramycin by fermentation, it is characterized in that, by kabicidin streptomycete (Streptomyces fungicidious) FYFJ03 constant temperature culture in fermention medium, to ferment by inoculation on solid slant culture base, fermentation condition is 25-30 ℃ of constant temperature culture, at fermenting process different steps stream, add different sugared concentration, improve the dissolved oxygen in fermented liquid, improve the fermentation level of enramycin; The sugar that stream adds is glucose, maltose, dextrin or other amylum hydrolysate of the sugar; In fermenting process, after fermentation 36～60 hours, stream adds the sugar that concentration is 200～300g/L; After fermentation 60～90 hours, stream adds the sugar that concentration is 350～450g/L; After fermentation 90 hours, stream adds the sugar that concentration is 500～600g/L.
2. the method for claim 1, is characterized in that, in fermenting process, after fermentation 36～60 hours, stream adds the sugar that concentration is 200～275g/L; After fermentation 60～90 hours, stream adds the sugar that concentration is 375～425g/L; After fermentation 90 hours, stream adds the sugar that concentration is 525～575g/L.
3. the method for claim 1, is characterized in that, in fermenting process, after fermentation 36～60 hours, stream adds the sugar that concentration is 250g/L; After fermentation 60～90 hours, stream adds the sugar that concentration is 400g/L; After fermentation 90 hours, stream adds the sugar that concentration is 550g/L.
4. the method as described in claim 1-3 any one, is characterized in that, 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.
5. the method as described in claim 1-3 any one, is characterized in that, 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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Cited By (2)
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| CN105506040A (en) * | 2015-12-23 | 2016-04-20 | 安徽丰原发酵技术工程研究有限公司 | Method for fermentation production of enramycin |
| CN106148460A (en) * | 2015-04-27 | 2016-11-23 | 牡丹江佰佳信生物科技有限公司 | A kind of fermentation medium improving enramycin B component yield and fermentation process |
Families Citing this family (3)
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| CN104195203B (en) * | 2014-08-29 | 2017-09-12 | 宁夏泰瑞制药股份有限公司 | A kind of antifungal streptomycete fermentation produces the culture medium and feed process of enramycin |
| CN104434777B (en) * | 2014-12-29 | 2015-10-21 | 江西兴鼎科技有限公司 | A kind of method improving enramycin premix finished product stability |
| CN105779535A (en) * | 2016-05-16 | 2016-07-20 | 新疆天富阳光生物科技有限公司 | Culture medium for fermenting and producing enramycin and fermentation method |
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