CN1899599A - Method for preparing Nosiheptide premixed agent - Google Patents
Method for preparing Nosiheptide premixed agent Download PDFInfo
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- CN1899599A CN1899599A CN 200510027876 CN200510027876A CN1899599A CN 1899599 A CN1899599 A CN 1899599A CN 200510027876 CN200510027876 CN 200510027876 CN 200510027876 A CN200510027876 A CN 200510027876A CN 1899599 A CN1899599 A CN 1899599A
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Abstract
The present invention belongs to the field of biomedicine preparing technology, relates to preparation of feed medicine additive, and is especially preparation process of pre-mixed preparation containing Nosiheptide as one kind of sulfur-containing polypeptide. The pre-mixed preparation containing Nosiheptide is prepared through the steps of preparing Nosiheptide generating microbe by means of gene engineering technology, Nosiheptide fermentation, separating extraction and adding supplementary material, stepped dilution, and mixing. The preparation process has raised oxygen utilizing efficiency of streptomycete, raised Nosiheptide yield and lowered production cost.
Description
Technical field
The invention belongs to field of biological pharmacy, relate to a kind of preparation method of medical feed additive, be specifically related to a kind of method for preparing sulfur-bearing polypeptide antibiotics Nosiheptide premixed agent.
Background technology:
Feed safety is the basic point of environmental protection food, and China is vast in territory, and is populous, and along with the paces of reform and opening-up are accelerated, living standards of the people improve, and the demand of beasts, birds and aquatic products is also increasing.Therefore China is a large agricultural country in addition, and the livestock product export volume is very big, but the drug residue problem is very serious, and it is very necessary to ensureing people's health and development China agricultural economy to research and develop green feed additive.July calendar year 2001, the Ministry of Agriculture issued the notice of " feedstuff medicated premix operating specification ", and had listed the feedstuff medicated premix of 33 kinds of compliant, and nosiheptide is one of them.It is a kind of novel non-absorptive-type feeding antibiotic.
New veterinary drug nosiheptide (has another name called nosiheptide, English name Nosiheptide) is bionic product, produces, be the sulfur-bearing polypeptide antibiotics by Streptomyces actuosus fermentation, its structure is 12 seed amino acids and the synthetic polypeptide of derivant, and molecular weight is 1222.Nosiheptide is a kind of yellow crystal, is insoluble in water.Its structure (Merck index, fourth edtion) is seen Fig. 7.Be made up of five thiazole rings, a pyridine ring, an indole ring, a threonine and a glutamic acid in the molecule, side chain is a dehydroalanine.Nosiheptide has very strong inhibitory action to gram positive bacteria, and its mechanism of action is elongation factor Tu and the G that suppresses in the translation process, suppresses the growth of gram positive bacteria.Suppress the synthetic of guanosine three, four phosphatases simultaneously.
Nosiheptide is external 1961 chemical compounds of finding, the seventies, France Lang Puluoke company was once studied it, be not developed to novel non-absorptive-type feeding antibiotic by MIT the eighties, and be legal feed additive by Japanese government's approval in December in 1987 25 days, listed Japanese feed safety method in, its commodity are called PrilnofaX (Hess; Clark).Because its not residual in vivo character is extensive use of by states such as the European Community, North America, Japan at present
Nosiheptide has following characteristics as feedstuff with additive: 1, and the animal specific antibiotic does not cause crossing drug resistant.2, not residual in animal body, contaminated food products not.3, addition few (5-10ppm), the growth promotion effect is (weightening finish 8-10%) obviously.And the raising price of deed.4. applied range can be used for fowl, poultry, fish etc.
Shanghai Medical Univ developed research to nosiheptide in 1988, obtained the Ministry of Agriculture in June, 1998 and issued the new veterinary drug certificate of nosiheptide (three classes) (crude drug and pre-mixing agent).And through China Veterinary Drugs Supervisory Inst. the Test Identification to the nosiheptide sample, product quality is in full accord with external like product.2002, Zhejiang Hangzhou remittance energy biotech company and Fudan University's cooperation were with the nosiheptide industrialization, and wherein, nosiheptide produces bacterium S.actuosus Z13, is to produce strain, and commodity are called " promise farming ".Year is sold the output value 7,000 ten thousand at present.And extensive use on fowl, poultry and Aquatic product, obtain good social benefit.But take a broad view of domestic and foreign literature, the report of the relevant Nosiheptide premixed agent preparation method of Shang Weijian.
Summary of the invention
Purpose of the present invention provides a kind of method for preparing Nosiheptide premixed agent.Especially the method for preparing sulfur-bearing polypeptide antibiotics Nosiheptide premixed agent.
The inventive method comprises the steps:
1, genetic engineering prepares nosiheptide and produces bacterium Streptomyces actuosus
The present invention produces at known nosiheptide on the research basis of bacterium S.actuosusZ13, adopts technique for gene engineering to change the metabolism that nosiheptide produces bacterium Z13, improves its output.
At first, adopt the DNA recombinant technique, structure contains the recombinant DNA of the gene vhb of Vitreoscilla hemoglobin (VHb), and (accompanying drawing 1 is seen in genetic engineering operation, 2), by transforming and changing Z13 in conjunction with the recombinant DNA that transfer method will contain the vhb gene, obtain engineering streptomycete Streptomyces actuosus, (engineering bacteria code name Z14, Z15).Under the insufficient situation of commercial production oxygen supply, because the proteic great expression of VHb has improved and enlivened streptomycete Streptomyces.actuosus Z14, Z15 is to the utilization ratio of oxygen, make thalli growth vigorous, thereby reach the purpose that improves the nosiheptide productive rate and reduce production costs.
Second, the present invention adopts technique for gene engineering, structure contains the recombinant DNA of the gene sam of S-adenosylmethionine synzyme SAMs, and (accompanying drawing 3 is seen in genetic engineering operation, 4), by transforming and changing Z13 in conjunction with the recombinant DNA that transfer method will contain the sam gene, acquisition engineering streptomycete Streptomyces actuosus engineering bacteria (engineering bacteria code name Z16, Z17).By the proteic expression of SAMs, changed the sulfo-that enlivens streptomycete Streptomyces.actuosus Z13 and thanked, improve the output of S-adenosylmethionine, thereby reached the purpose that improves nosiheptide output.
The 3rd, the present invention adopts technique for gene engineering to make up the recombinant DNA that contains vhb and two genes of sam, (accompanying drawing 5 is seen in the genetic engineering operation, 6), by transforming and changing Z13 in conjunction with the recombinant DNA that transfer method will contain vhb and two genes of sam, obtain engineering streptomycete Streptomyces actuosus (engineering bacteria code name Z18, Z19), enliven the metabolism of streptomycete by VHb and the proteic expression change of SAMs, reach the purpose that improves nosiheptide output.
2. nosiheptide fermentation;
The nosiheptide fermentation medium is made up of analysis for soybean powder, glucose, inorganic salt etc., and the control optimum temperature is 26-30 ℃, and pH is neutral, and fermentation period is 9-10 days.
3, the separation and Extraction nosiheptide:
Adopt the solvent method that endobacillary nosiheptide is extracted, fermentation liquid is measured the volume number, adds equal-volume ethanol, homogenate, centrifugal, collect supernatant, concentrate, with n-butanol extraction, concentrate evaporate to dryness, add the dioxane dissolving, add the water precipitation nosiheptide again, sedimentary nosiheptide washes with water, and the oven dry.Measure content and press veterinary drug allusion quotation detection of biological activity with HPLC.
4 preparation Nosiheptide premixed agents:
Get nosiheptide, add starch, soybean cake powder, bran powder, Semen Maydis powder, wheat bran, rice protein powder, stone powder (CaCO
3), adjuvant such as zeolite powder, stepwise dilution mixes the pre-mixing agent of mixing, make the 0.25%-10% different size thoroughly.Measure content and press pharmacopeia detection of biological activity with HPLC.
Description of drawings
Figure .1vhb transforms and expresses plasmid construction
Fig. 2 contains the structure of vhb integration vector
Figure .3 contains sam and transforms the expression plasmid construction
Fig. 4 contains the structure of sam integration vector
Fig. 5 contains the structure of vhb and sam conversion carrier
Fig. 6 contains the structure of vhb and sam integration vector
Fig. 7, the nosiheptide structure chart.
The specific embodiment
Nosiheptide produces bacterium Z13 and is inoculated in seed tank, grew 40-48 hour, insert 30 tons of fermentation tanks by 5% inoculum concentration, with 1: 1 ventilation, the control temperature is 28-30 ℃, stirs 160-180 rev/min wherein according to pH and sugared content, thalli morphology feed supplement, cultivates 9 days, 20 tons of dischargings, every liter of fermentation unit reaches 2.3 grams.Obtain 23.46 kilograms of nosiheptide (purity 85%) through extraction; Add starch, soybean cake powder, bran powder, Semen Maydis powder, wheat bran, rice protein powder, stone powder (CaCO
3), adjuvant such as zeolite powder, stepwise dilution, mixing is mixed thoroughly, 7.98 tons of pre-mixing agents (0.25% specification).
Nosiheptide produces bacterium Z14 and is inoculated in seed tank, grew 40-48 hour, insert 30 tons of fermentation tanks by 5% inoculum concentration, with 1: 0.5 ventilation, the control temperature was 28-30 ℃, stirs 160-180 rev/min, wherein according to pH and sugared content, thalli morphology feed supplement, cultivated 9 days, 20 tons of dischargings, every liter of fermentation unit reaches 3.2 grams.Obtain 32.6 kilograms of nosiheptide (purity 85%) through extraction; Add starch, soybean cake powder, wheat bran, bran powder, rice protein powder, stone powder (CaCO
3), zeolite powder etc., stepwise dilution, mixing is mixed thoroughly, 11.08 tons of pre-mixing agents (0.25% specification).
Nosiheptide produces bacterium Z15 and is inoculated in seed tank, grew 40-48 hour, insert 30 tons of fermentation tanks by 5% inoculum concentration, with 1: 0.5 ventilation, the control temperature was 28-30 ℃, stirs 160-180 rev/min, wherein according to pH and sugared content, thalli morphology feed supplement, cultivated 9 days, 21 tons of dischargings, every liter of fermentation unit reaches 4.2 grams.Obtain 44.982 kilograms of nosiheptide (purity 85%) through extraction; Add starch, soybean cake powder, bran powder, wheat bran, Semen Maydis powder, stone powder (CaCO
3), zeolite powder etc., stepwise dilution, mixing is mixed thoroughly, 7.65 tons of pre-mixing agents (0.5% specification).
Nosiheptide produces bacterium Z16 and is inoculated in seed tank, grew 40-48 hour, insert 50 tons of fermentation tanks by 5% inoculum concentration, with 1: 0.8 ventilation, the control temperature was 28-30 ℃, stirs 160 rev/mins, wherein according to pH and sugared content, thalli morphology feed supplement, cultivated 9 days, 35 tons of dischargings, every liter of fermentation unit reaches 4.5 grams.Obtain 80.33 kilograms of nosiheptide (purity 85%) through extraction; Add bran powder, wheat bran, Semen Maydis powder, rice protein powder, stone powder (CaCO
3), zeolite powder etc., stepwise dilution, mixing is mixed thoroughly, 13.656 tons of pre-mixing agents (0.5% specification).
Nosiheptide produces bacterium Z19 and is inoculated in seed tank, grew 40-48 hour, insert 50 tons of fermentation tanks by 5% inoculum concentration, with 1: 0.5 ventilation, the control temperature was 28-30 ℃, stirs 160 rev/mins, wherein according to pH and sugared content, thalli morphology feed supplement, cultivated 9 days, 35 tons of dischargings, every liter of fermentation unit reaches 5.0 grams.Obtain 89.25 kilograms of nosiheptide (purity 85%) through extraction; Add bran powder, wheat bran, Semen Maydis powder, rice protein powder, stone powder (CaCO
3), zeolite powder etc., stepwise dilution, mixing is mixed thoroughly, 1.517 tons of pre-mixing agents (5% specification).
Claims (5)
1, a kind of method for preparing Nosiheptide premixed agent is characterized in that comprising the steps:
1) genetic engineering prepares nosiheptide generation bacterium;
2) nosiheptide fermentation;
3) separation and Extraction nosiheptide;
4) get above-mentioned nosiheptide, add adjuvant, stepwise dilution mixes and mixes, prepares Nosiheptide premixed agent thoroughly.
2, by the described method for preparing Nosiheptide premixed agent of claim 1, it is characterized in that adopting in the described step 1 the DNA recombinant technique, structure contains the recombinant DNA of the gene vhb of Vitreoscilla hemoglobin, by transforming and changing Z13 in conjunction with shifting the recombinant DNA that will contain the vhb gene, obtain engineering streptomycete Z14, Z15;
Structure contains the recombinant DNA of the gene sam of S-adenosylmethionine synzyme SAMs, by transforming and changing Z13 in conjunction with shifting the recombinant DNA that will contain the sam gene, obtains engineering streptomycete Z16, Z17;
Build the recombinant DNA that contains vhb and two genes of sam,, obtain engineering streptomycete Z18, Z19 by transforming and changing Z13 in conjunction with shifting the recombinant DNA that will contain vhb and two genes of sam.
3, by the described method for preparing Nosiheptide premixed agent of claim 1, it is characterized in that described step 2 wherein the nosiheptide fermentation medium contain analysis for soybean powder, glucose and inorganic salt composition, the control temperature is 26-30 ℃, pH be a neutrality, fermentation period is 9-10 days.
4, by the described method for preparing Nosiheptide premixed agent of claim 1, it is characterized in that described step 3 wherein adopts the solvent method to extract endobacillary nosiheptide, fermentation liquid is measured the volume number, add equal-volume ethanol, homogenate, centrifugal, collect supernatant, concentrate, with n-butanol extraction, concentrate evaporate to dryness, add the dioxane dissolving, add the water precipitation nosiheptide again, the water washing and precipitating thing, content and detection of biological activity are measured in oven dry.
5, by the described method for preparing Nosiheptide premixed agent of claim 1, it is characterized in that described step 4 wherein the content of nosiheptide be 0.25%-10%, described adjuvant comprises starch, soybean cake powder, bran powder, Semen Maydis powder, wheat bran, rice protein powder, CaCO
3And zeolite powder.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101869188A (en) * | 2010-06-11 | 2010-10-27 | 濮阳泓天威药业有限公司 | Preparation method of nosiheptide premix |
| CN101303334B (en) * | 2008-06-06 | 2011-08-24 | 浙江汇能动物药品有限公司 | HPLC testing method of Nosiheptide product biological valence content |
| CN102210630A (en) * | 2011-06-02 | 2011-10-12 | 浙江汇能动物药品有限公司 | System and method for preparing nosiheptide premix particles |
| CN101624418B (en) * | 2009-08-03 | 2012-04-18 | 安徽省皖北药业股份有限公司 | Method for preparing nosiheptide powder |
| CN104719634A (en) * | 2015-02-10 | 2015-06-24 | 河北圣雪大成制药有限责任公司 | Method for preparing nosiheptide pre-mixing agent |
| CN105126073A (en) * | 2015-09-02 | 2015-12-09 | 浙江汇能动物药品有限公司 | High-stability nosiheptide solution and preparation method thereof |
| CN105198958A (en) * | 2015-09-29 | 2015-12-30 | 浙江汇能动物药品有限公司 | Nosiheptide finemeal extracting method |
| CN105695351A (en) * | 2015-12-29 | 2016-06-22 | 西藏天虹科技股份有限责任公司 | Streptomyces actuosus LB-16 and method for preparing nosiheptide from same |
| CN110575532A (en) * | 2018-06-08 | 2019-12-17 | 上海莫息生物科技有限公司 | Nosiheptide soluble powder and preparation method thereof |
-
2005
- 2005-07-19 CN CN 200510027876 patent/CN1899599A/en active Pending
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101303334B (en) * | 2008-06-06 | 2011-08-24 | 浙江汇能动物药品有限公司 | HPLC testing method of Nosiheptide product biological valence content |
| CN101624418B (en) * | 2009-08-03 | 2012-04-18 | 安徽省皖北药业股份有限公司 | Method for preparing nosiheptide powder |
| CN101869188A (en) * | 2010-06-11 | 2010-10-27 | 濮阳泓天威药业有限公司 | Preparation method of nosiheptide premix |
| CN102210630A (en) * | 2011-06-02 | 2011-10-12 | 浙江汇能动物药品有限公司 | System and method for preparing nosiheptide premix particles |
| CN102210630B (en) * | 2011-06-02 | 2014-01-15 | 浙江汇能动物药品有限公司 | System and method for preparing nosiheptide premix particles |
| CN104719634B (en) * | 2015-02-10 | 2018-08-24 | 河北圣雪大成制药有限责任公司 | A method of preparing Nosiheptide premixed agent |
| CN104719634A (en) * | 2015-02-10 | 2015-06-24 | 河北圣雪大成制药有限责任公司 | Method for preparing nosiheptide pre-mixing agent |
| CN105126073A (en) * | 2015-09-02 | 2015-12-09 | 浙江汇能动物药品有限公司 | High-stability nosiheptide solution and preparation method thereof |
| CN105126073B (en) * | 2015-09-02 | 2019-04-05 | 浙江汇能生物股份有限公司 | A kind of high stability Nosiheptide solution and preparation method thereof |
| CN105198958A (en) * | 2015-09-29 | 2015-12-30 | 浙江汇能动物药品有限公司 | Nosiheptide finemeal extracting method |
| CN105695351A (en) * | 2015-12-29 | 2016-06-22 | 西藏天虹科技股份有限责任公司 | Streptomyces actuosus LB-16 and method for preparing nosiheptide from same |
| CN105695351B (en) * | 2015-12-29 | 2019-04-09 | 西藏天虹科技股份有限责任公司 | S.actuosus LB-16 and the method for preparing Nosiheptide using it |
| CN110575532A (en) * | 2018-06-08 | 2019-12-17 | 上海莫息生物科技有限公司 | Nosiheptide soluble powder and preparation method thereof |
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Open date: 20070124 |