CN101245362B - Method for producing polypeptide enramycin with zymotechnics - Google Patents
Method for producing polypeptide enramycin with zymotechnics Download PDFInfo
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- 239000008103 glucose Substances 0.000 claims abstract description 36
- 238000000034 method Methods 0.000 claims abstract description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 241000276851 Streptomyces sp. NJWGY3665 Species 0.000 claims abstract description 23
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
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- 235000016709 nutrition Nutrition 0.000 claims description 9
- 229910052700 potassium Inorganic materials 0.000 claims description 8
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- 229910019142 PO4 Inorganic materials 0.000 claims description 6
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- 108010080698 Peptones Proteins 0.000 claims description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 6
- 229920002472 Starch Polymers 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
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- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 5
- 241001655322 Streptomycetales Species 0.000 claims description 4
- 238000011534 incubation Methods 0.000 claims description 3
- 244000131316 Panax pseudoginseng Species 0.000 claims description 2
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 claims description 2
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- NJCUSQKMYNTYOW-MWUYRYRWSA-N enramicina Chemical compound O.N1C(=O)NC(=O)C(C=2C=C(Cl)C(O)=C(Cl)C=2)NC(=O)C(CO)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(CC2N=C(N)NC2)NC(=O)C(CCCNC(N)=O)NC(=O)C(C(C)O)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C(C)O)NC(=O)N(CCCCN)C(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)/C=C/C=C/CCCCC(C)CC)C(C)OC(=O)C(C=2C=CC(O)=CC=2)NC(=O)C(C)NC(=O)C1CC1CNC(N)=N1 NJCUSQKMYNTYOW-MWUYRYRWSA-N 0.000 abstract description 28
- 229950003984 enramycin Drugs 0.000 abstract description 28
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a method for producing polypeptide antibiotics enramycin by the fermentation method, which pertains to the fields of agricultural biotechnology, industrial biotechnology and the fermentation engineering. The method is as follows: (1) slant culture: a Streptomyces sp.NJWGY3665 bacterial strain is inoculated in a glucose nutrient culture medium to carry out the slant culture, the culture temperature is 28 to 37 DEG C and the culture time is 2 to 6 days; (2) seed culture: a spore which is cultured on a slant is produced into a single-spore suspension by using sterile water, which is also inoculated in a seed culture medium for culture, the temperature is 28 to 32 DEG C, the rotational speed is 200rpm and the culture lasts for 2 to 6 days; (3) fermentation culture: the seed liquid is inoculated in a fermentation culture medium for culture, the temperature is controlled at 28 to 37 DEG C, the pH is controlled at 6.0 to 9.0, the rotational speed is 180 to 220rpm, the fermentation lasts for 5 to 9 days, so as to obtain the Streptomyces sp., methanol or ethanol is used for the extraction and separation of mycelium through the method of centrifugal separation, then the resin method is adopted for carrying out refining, so as to obtain the peptide antibiotics enramycin.
Description
Technical field
What the present invention relates to is a kind of method of producing polypeptide enramycin with zymotechnics, belongs to Agricultural biotechnologies, industrial biotechnology, field of fermentation engineering.
Background technology
Microbiotic makes an addition in the feed in order to promote animal growth and development and to improve the existing nearly 60 years history of feed report that many countries are the prolonged application Antibiotic Additive all.Yet, being accompanied by being extensive use of of antibiotics growth promoter, a series of problems also appear gradually, mainly are drug residue and resistance problem.For a long time, extensive, indiscriminately use antibiotic feed to have brought great trouble and difficulty for the control of bacteriosis and treatment, also bring potential to threaten simultaneously to human beings'health.Though, facts have proved at present about whether should in feed, using microbiotic to have numerous disputes, reasonably use Antibiotic Additive can promote growth of animals or poultry, improve food conversion ratio.Also there is simultaneously report to show, forbids that the country that adds microbiotic or other antibiotic growth promoters in feed can cause the increase of livestock industry production cost, profit to reduce, and also might cause such as the problems such as trade war between ecotope and the country simultaneously.In fact, Antibiotic Additive has only they comprehensively is familiar with and reasonably application still in a large amount of uses, just can give full play to its advantageous effect, and avoid human health is worked the mischief.The task of top priority is the resistance at microorganism, research and development wide spectrum, efficient, safe novel fodder additive, and this can not only bring remarkable economic efficiency, also has extremely important social effect.
Enramycin (Enramycin) is a kind of wide spectrum, efficient, safe novel fodder additive.It has another name called grace bleomycin, grace mycin, enramycin, is a kind of polypeptide antibiotics that is produced by streptomycete fermentation.As a kind of brand-new feed specialist additive, have following characteristics: the enramycin that 1. adds trace in the feed just can promote the weightening finish to livestock and poultry.2. have intensive antimicrobial acivity, can suppress the harmful bacterium (clostridium) in the intestines especially energetically gram-positive microorganism.3. because absorptivity not, only its effect of performance in alimentary canal can not residue in the livestock and poultry body.4. and between existing clinically microbiotic or the antimicrobial drug there is not cross resistance.Sensitive organism produces resistance to enramycin hardly, and under experiment condition, chemical sproof generation is also very slow, even occur, resistance is also unstable, and very easily loses.Therefore the long-term continuous time spent, its effect can not be affected yet.5. satisfactory stability is arranged in feed.In addition, enramycin also has hepatitis B virus (HBV) antigen and hepatitis B virus e antigen (HBeAg) and suppresses effect preferably.Just because of so many advantage is arranged, enramycin has become a line products of antibiotic feed additive for promoting growth.
Scholarly forecast, as a kind of animal specific microbiotic, enramycin also can serve as the leading role of growth promoter in considerable time, because it both can satisfy the requirement of people to security, can significantly reduce the animal product cost again.But present enramycin complex technical process, fermentation time is long, the production cost height, unit tires low.
Summary of the invention
The objective of the invention is at above-mentioned weak point, screen a strain enramycin and produced bacterium, and provide a kind of method of producing polypeptide enramycin with zymotechnics, according in the strain growth metabolic process to the difference of oxygen demand, control dissolved oxygen stage by stage, this is integrated fermentations of multistage such as an aerobic, little oxygen, and adopts stream to add the method for carbon source, has realized the high-density culture of enramycin production bacterium streptomycete.The method technological process of producing polypeptide enramycin with zymotechnics of the present invention is simple, and production cost is low, the enramycin height of tiring in the unit volume fermented liquid.
Adopt streptomycete Streptomyces sp.NJWGY3665 in the method for producing polypeptide enramycin with zymotechnics of the present invention, this bacterium adds nutritive substances such as glucose by stream in initial low glucose concentrations (1%) fermentation, production process, and according to having realized high efficient fermentation under the situation of fermentation different steps to the demand difference of dissolved oxygen, enramycin is tired and is reached 6100 μ g/mL in the unit volume fermented liquid.
Streptomycete Streptomyces sp.NJWGY3665 bacterial strain is to separate to obtain from the soil of school district, Nanjing University of Technology Jiangpu.This streptomyces species is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center at present, registering on the books and number in this preservation center is CGMCCNO.2410, classification name: streptomycete, the Latin formal name used at school of bacterial classification is Streptomyces sp., the microorganism (strain) of ginseng certificate: NJWGY3665, preservation date are on March 21st, 2008.
The method of producing polypeptide enramycin with zymotechnics is to take following mode to realize:
The method of producing polypeptide enramycin with zymotechnics is:
1, slant culture: streptomycete Streptomyces sp.NJWGY3665 bacterial classification is received in the glucose nutritional medium, carry out slant culture, the weight percent proportioning of glucose nutritional medium is glucose 0.5～1%, extractum carnis 0.5～1%, peptone 0.5～1%, NaCl 0.3～0.5% or K
2HPO
40.1～0.2%, agar 1.5～2%, water surplus, pH 6.0-9.0, culture temperature 28-37 ℃, incubation time 2 days-6 days;
2, seed culture: with sterilized water the spore of cultivating on the inclined-plane is made monospore suspension, and be inoculated in the 50ml seed culture medium and cultivate, the weight percent proportioning of seed culture medium is glucose 1～3.0%, corn steep liquor 1～3%, CaCO
31～2%, yeast powder 0.5%, NaCl 0.1～0.8%, water surplus, pH is 6.0-9.0, and temperature is 28～32 ℃, and rotating speed is 200rpm, cultivates 2 days-6 days;
3, fermentation culture: seed liquor is inoculated in the fermention medium cultivates, the weight percent proportioning of fermention medium is glucose 2.0-5.0%, starch 0.5-2.0%, corn steep liquor 0.5-3.0%, yeast extract paste 0.1～0.5%, soybean cake powder 0.5～2%, NaCl 0.5-1.0%, CaCO
31～2%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, water surplus, temperature is controlled at 28-37 ℃, pH is controlled at 6.0-9.0, rotating speed 180-220rpm, fermented 5-9 days, and obtained streptomycete Streptomyces sp.NJWGY3665, adopt the method for centrifugation that mycelium is carried out extraction separation with methyl alcohol or ethanol, adopt the ion exchange resin method to make with extra care separation again, obtain polypeptide enramycin.
Adopt HPLC to carry out quantitative analysis, can learn the concentration of enramycin in the sample.Enramycin is 3630 μ g/ml～6630 μ g/ml in the unit volume fermented liquid.
Streptomycete Streptomyces sp.NJWGY3665 is after cultivating on the glucose nutrient agar plate, and a large amount of colourless vegetative myceliums are tiled in media surface, and aerial hyphae presents white to little grey.
Adopt streptomycete Streptomyces sp.NJWGY3665 in the method for producing polypeptide enramycin with zymotechnics of the present invention, this bacterium is being realized high efficiency conversion, and the enramycin productive rate is 6630 μ g/mL.Streptomycete Streptomyces sp.NJWGY3665 bacterial strain is the bacterial strain that produces antimicrobial substance through the strain that autonomous screening and separating arrives, and its tunning enramycin has the intensive antimicrobial acivity, and sensitive organism produces resistance to enramycin hardly.The method technological process of producing polypeptide enramycin with zymotechnics of the present invention is simple, and production cost is low, the unit height of tiring.
The polypeptide enramycin that the method for producing polypeptide enramycin with zymotechnics of the present invention is produced can be used as a kind of wide spectrum, efficient, safe novel fodder additive.
Embodiment
Embodiment 1:
Streptomycete Streptomyces sp.NJWGY3665 bacterial classification is received glucose 0.5%, extractum carnis 0.5%, peptone 0.5%, NaCl 0.5%, and agar 1.5%, water 96.5% are in the glucose nutritional medium of pH6.0, carry out slant culture, cultivated 2 days down at 28 ℃.Get slant strains, add 2-3ml sterilized water (the test tube slant bacterial classification of per 18 * 180mm), gently lawn is washed, make spore suspension, get the 0.5ml spore suspension and be inoculated into and contain 40ml glucose 1.0%, corn steep liquor 3%, CaCO with transfering loop
31%, yeast extract paste 0.5%, NaCl 0.1%, water 94.4% were cultivated 3 days in the liquid seed culture medium of pH7.0, and culture temperature is 28 ℃, and rotating speed is 200rpm.The 500ml that the 5m1 seed liquor is linked into liquid amount again and is the 100ml fermention medium shakes in the bottle, at 28 ℃, 180rpm, shaker fermentation was cultivated 5 days under the condition of pH6.0, obtain streptomycete Streptomyces sp.NJWGY3665, adopt the method for centrifugation that mycelium is carried out extraction separation with methyl alcohol or ethanol, adopt the ion exchange resin method to make with extra care separation again, obtain polypeptide enramycin.
Adopt HPLC to carry out quantitative analysis, can learn the concentration of enramycin in the sample.Enramycin is 3630 μ g/ml in the unit volume fermented liquid.
The proportioning of described fermention medium is a glucose 4.0%, starch 2.0%, corn steep liquor 2.0%, yeast extract paste 0.1%, soybean cake powder 2%, NaCl 0.5%, CaCO
31.5%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, water 86.9%.
Streptomycete Streptomyces sp.NJWGY3665 is after cultivating on the glucose nutrient agar plate, and a large amount of colourless vegetative myceliums are tiled in media surface, and aerial hyphae presents white to little grey.
Embodiment 2:
Streptomycete Streptomyces sp.NJWGY3665 bacterial classification is received glucose 0.5%, extractum carnis 0.8%, peptone 0.8%, K
2HPO
40.1%, agar 1.8%, water 96% in the glucose nutritional medium of pH7.0, carry out slant culture, cultivate 3 days down at 37 ℃.Get slant strains, add 2-3ml sterilized water (the test tube slant bacterial classification of per 18 * 180mm), gently lawn is washed with transfering loop, make spore suspension, get the 0.5ml spore suspension and be inoculated into spore suspension and be inoculated into and contain 40ml glucose 2.0%, corn steep liquor 2.0%, CaCO
31.5%, yeast powder 0.5%, NaCl 0.5%, water 93.5% were cultivated 4 days in the liquid seed culture medium of pH7.0, and culture temperature is 30 ℃, and rotating speed is 200rpm.The 500ml that the 10ml seed liquor is linked into liquid amount again and is the 100ml fermention medium shakes in the bottle, at 32 ℃, 220rpm, shaker fermentation was cultivated 6 days under the condition of pH7.0, obtain streptomycete Streptomyces sp.NJWGY3665, the method that adopts centrifugation is carried out extraction separation with mycelium with ethanol, adopt the ion exchange resin method to make with extra care separation again, obtain polypeptide enramycin.
Adopt HPLC to carry out quantitative analysis, can learn the concentration of enramycin in the sample.Enramycin is 4630 μ g/ml in the unit volume fermented liquid.
The proportioning of described fermention medium is a glucose 2.0%, starch 1.5%, corn steep liquor 0.5%, yeast extract paste 0.3%, soybean cake powder 1.5%, NaCl 0.8%, CaCO
31.5%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, water 90.9%.
Streptomycete Streptomyces sp.NJWGY3665 is after cultivating on the glucose nutrient agar plate, and a large amount of colourless vegetative myceliums are tiled in media surface, and aerial hyphae presents white to little grey.
Embodiment 3:
Adopt broth culture streptomycete Streptomyces sp.NJWGY3665 bacterial classification to be received glucose 1%, extractum carnis 1%, peptone 1%, NaCl 0.5%, and agar 2%, water 94.5% are in the glucose nutritional medium of pH9.0, carry out slant culture, cultivated 5 days down at 32 ℃.Get slant strains, add 2-3ml sterilized water (the test tube slant bacterial classification of per 18 * 180mm), gently lawn is washed, make spore suspension, get the 1ml spore suspension and be inoculated into and contain 50ml glucose 3.0%, corn steep liquor 1%, CaCO with transfering loop
32%, yeast powder 0.5%, NaCl 0.8%, water 92.7% were cultivated 4 days in the liquid seed culture medium of pH 9.0, and culture temperature is 32 ℃, and rotating speed is 200rpm.Just the 50ml seed liquor is inoculated in the 2L fermentor tank that liquid amount is the 1.5L fermention medium again, and at 37 ℃, air is that 1.0L/min flows into the overall flow rate, and mixes with 400rpm, keeps oxyty (DO) and is 60-100%.With 20% (w/v) NaOH the pH value is maintained 8.5-9.0, appropriate supplement sugar, when cell concn optical density(OD) (O.D.) when level reaches 32, change little oxygen over to, keep oxyty (DO) and be 5-10%, fermentation culture 9 days, obtain streptomycete Streptomyces sp.NJWGY3665, the method that adopts centrifugation is carried out extraction separation with mycelium with ethanol, adopt resin to make with extra care again, obtain polypeptide enramycin.
Adopt HPLC to carry out quantitative analysis, can learn the concentration of enramycin in the sample.Enramycin is 5630 μ g/ml in the unit volume fermented liquid.
The proportioning of described fermention medium is a glucose 5.0%, starch 0.5%, corn steep liquor 3.0%, yeast extract paste 0.5%, soybean cake powder 0.5%, NaCl 1.5%, CaCO
31.5%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, water 95%.
Streptomycete Streptomyces sp.NJWGY3665 is after cultivating on the glucose nutrient agar medium eggplant bottle, and a large amount of colourless vegetative myceliums are tiled in media surface, and aerial hyphae presents white to little grey.
Embodiment 4:
Adopt broth culture streptomycete Streptomyces sp.NJWGY3665 bacterial classification to be received glucose 0.8%, extractum carnis 1%, peptone 1%, K
2HPO
40.2%, agar 2%, water 95% in the glucose nutritional medium of pH 8.0, carry out slant culture, cultivate 6 days down at 37 ℃.Get slant strains, add 2-3ml sterilized water (the test tube slant bacterial classification of per 18 * 180mm), gently lawn is washed with transfering loop, make spore suspension, get the 0.5ml spore suspension and be inoculated into spore suspension and be inoculated into and contain 50ml glucose 3.0%, corn steep liquor 3.0%, CaCO
32%, yeast powder 0.5%, NaCl 0.8%, water 90.7% are cultivated 48h in the liquid seed culture medium of pH9.0, and culture temperature is 32 ℃, and rotating speed is 200rpm.The 50ml seed liquor is inoculated in the 2L fermentor tank that liquid amount is the 1.5L fermention medium, and at 33 ℃, air is that 1.5L/min flows into the overall flow rate, and mixes with 350rpm, keeps oxyty (DO) and is 60-100%.20% (w/v) NaOH maintains 8.0-8.5 with the pH value, stream adds nutrition such as glucose, when cell concn optical density(OD) (O.D.) when level reaches 38, change little oxygen over to, keep oxyty (DO) and be 5-10%, fermentation culture 9 days, obtain streptomycete Streptomyces sp.NJWGY3665, the method that adopts centrifugation is carried out extraction separation with mycelium with ethanol, adopt the resin method to make with extra care again, obtain polypeptide enramycin.
Can adopt HPLC to carry out quantitative analysis, can learn the concentration of enramycin in the sample.Enramycin is 6630 μ g/ml in the unit volume fermented liquid.
The proportioning of described fermention medium is a glucose 5.0%, starch 2%, corn steep liquor 1.5%, yeast extract paste 0.5%, soybean cake powder 2%, NaCl 0.8%, CaCO
31.5%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, water 85.7%.
Streptomycete Streptomyces sp.NJWGY3665 is after cultivating on the glucose nutrient agar medium eggplant bottle, and a large amount of colourless vegetative myceliums are tiled in media surface, and aerial hyphae presents white to little grey.
Claims (2)
1. the method for a producing polypeptide enramycin with zymotechnics is characterized in that method is:
(1) slant culture: streptomycete Streptomyces sp.NJWGY3665 bacterial classification is received in the glucose nutritional medium, carry out slant culture, the weight percent proportioning of glucose nutritional medium is glucose 0.5～l%, extractum carnis 0.5～l%, peptone 0.5～l%, NaCl0.3～0.5% or K
2HPO
40.1～0.2%, agar 1.5～2%, water surplus, pH 6.0-9.0, culture temperature 28-37 ℃, incubation time 2 days-6 days;
(2) seed culture: with sterilized water the spore of cultivating on the inclined-plane is made monospore suspension, and be inoculated in the 40ml seed culture medium and cultivate, the weight percent proportioning of seed culture medium is glucose 1～3.0%, corn steep liquor 1～3%, CaCO
31～2%, yeast powder 0.5%, NaCl0.1～0.8%, water surplus, pH is 6.0-9.0, and temperature is 28～32 ℃, and rotating speed is 200rpm, incubation time 2 days-6 days;
(3) fermentation culture: seed liquor is inoculated in the fermention medium cultivates, the weight percent proportioning of fermention medium is glucose 2.0-5.0%, starch 0.5-2.0%, corn steep liquor 0.5-3.0%, yeast extract paste 0.1～0.5%, soybean cake powder 0.5～2%, NaCl 0.5-1.5%, CaCO
31.5%, NH
4Cl 0.5%, potassium primary phosphate 0.2%, ammonium sulfate 0.3%, water surplus, temperature is controlled at 28-37 ℃, pH is controlled at 6.0-9.0, rotating speed 180-220rpm, fermented 5-9 days, and obtained streptomycete Streptomyces sp.NJWGY3665, adopt the method for centrifugation that mycelium is carried out extraction separation with methyl alcohol or ethanol, adopt the resin method to make with extra care separation again, obtain polypeptide enramycin;
Described streptomyces species is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, registering on the books and number in this preservation center is CGMCC NO.2410, classification name: streptomycete, the Latin formal name used at school of bacterial classification is Streptomyces sp., the microorganism (strain) of ginseng certificate: NJWGY3665, preservation date are on March 21st, 2008.
2. the method for producing polypeptide enramycin with zymotechnics according to claim 1, it is characterized in that described streptomycete Streptomyces sp.NJWGY3665 is after cultivating on the glucose nutrient agar plate, a large amount of colourless vegetative myceliums are tiled in media surface, and aerial hyphae presents white to little grey.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101928701B (en) * | 2009-06-23 | 2012-07-04 | 华东理工大学 | Method for improving enzyme production and secondary metabolites of fungi |
| CN101899490B (en) * | 2010-07-14 | 2012-09-05 | 山东胜利股份有限公司 | Method for producing enramycin by using microbial fermentation |
| CN102154419A (en) * | 2010-12-20 | 2011-08-17 | 安徽丰原发酵技术工程研究有限公司 | Fermentation medium for improving enramycin yield |
| CN102174624B (en) * | 2010-12-31 | 2013-12-18 | 安徽丰原发酵技术工程研究有限公司 | Method for producing enramycin by fermentation |
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| CN102391363B (en) * | 2011-09-26 | 2013-11-13 | 浙江升华拜克生物股份有限公司 | Method for extracting enramycin |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1872854A (en) * | 2005-05-31 | 2006-12-06 | 西北农林科技大学农药研究所 | Antibiotic in lactam class, and prepartion method |
| WO2007068055A1 (en) * | 2005-12-15 | 2007-06-21 | Hancroft Pty Ltd | A method for preventing reduced feed intake in animals and treatment of disease conditions |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1872854A (en) * | 2005-05-31 | 2006-12-06 | 西北农林科技大学农药研究所 | Antibiotic in lactam class, and prepartion method |
| WO2007068055A1 (en) * | 2005-12-15 | 2007-06-21 | Hancroft Pty Ltd | A method for preventing reduced feed intake in animals and treatment of disease conditions |
Non-Patent Citations (2)
| Title |
|---|
| 卜仕金.多肽类抗生素饲料添加剂－安来霉素.《中国兽医杂志》.2003,第39卷(第4期),48-50. * |
| 浦宇等.吸附树脂及其在天然产物和抗生素中的应用.《中国医药工业杂志》.2003,第34卷(第12期),636-644. * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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