CN107619429A - A kind of Nosiheptide method of purification - Google Patents
A kind of Nosiheptide method of purification Download PDFInfo
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- CN107619429A CN107619429A CN201610560825.8A CN201610560825A CN107619429A CN 107619429 A CN107619429 A CN 107619429A CN 201610560825 A CN201610560825 A CN 201610560825A CN 107619429 A CN107619429 A CN 107619429A
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- nosiheptide
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Abstract
The present invention relates to a kind of Nosiheptide method of purification, extraction is carried out to the solid portion after Nosiheptide production fermentation using the mixing digestion agent of dichloromethane and methanol and obtains leaching liquor, remove the dichloromethane in leaching liquor and methanol obtains Nosiheptide crude extract, the Nosiheptide crude extract is washed with n-hexane to obtain the refined thing of Nosiheptide.Using method provided by the present invention, technical process present in existing Nosiheptide extraction process can be overcome numerous and diverse, production cycle length, the defects of production process disposal of pollutants is more.The efficiency of production process is improved, reduces the wasting of resources and environmental pollution, obtains the Nosiheptide product that purity is not less than 96.2%.
Description
Technical field
The invention belongs to chemical pharmacy field, and in particular to a kind of method of purification of veterinary drug feed addictive Nosiheptide.
Background technology
Nosiheptide is a kind of Thiopeptide antibiotics as caused by S.actuosus, because it is not easy to absorb in enteron aisle,
It can decompose after excreting and rapidly, so with noresidue, being not likely to produce drug resistance, being also resistance to without intersecting with other antibiotic
The features such as property of medicine, small effect on environment.Nosiheptide can be obviously promoted the growth of chicken, pig etc. and increase dressing percentage, at present by regarding
For a kind of environment-protecting feed additive of new non-absorbing type.
The purification preparation method of existing Nosiheptide mainly includes following several：
Alcohol solvent is extracted, and n-butanol is stripped (such as：Li Xia etc., the separation and Extraction of feed addictive Nosiheptide and refined,
Industrial microorganism, 1991,21 (1):19-22).Butanol extraction liquid thickening temperature is high in the method, and Nosiheptide is at high temperature
It is unstable, so bringing very big difficulty to extraction work；A large amount of emulsion layers are easily produced during extracting n-butyl alcohol, separation emulsion layer makes
The yield of product substantially reduces (crude product 70%, fine work 50%).
DMF (DMF) extracts, and distillation leaching liquor obtains concentrate, and then adds distillation water crystallization, most
After purify crude product.Prepared by the method, vapo(u)rizing temperature is high, and Nosiheptide easily decomposes；Crystallization is needed to prepare in preparation process, process route
It is cumbersome.
It can be seen that existing method of purification is difficult to the requirement for meeting industrialized production.It is necessary to provide a kind of technical process letter
Just, with short production cycle, environment-friendly industrialization Nosiheptide extraction process, to promote the development of Nosiheptide application field.
The content of the invention
It is an object of the invention to solve the above-mentioned problems in the prior art, there is provided a kind of production technology is easy, raw
Produce the Nosiheptide method of purification that the cycle is short, environment-friendly, yield is high and purity is good.
To achieve the above objectives, the invention provides a kind of Nosiheptide method of purification, using dichloromethane and methanol
Mixing digestion agent to Nosiheptide production fermentation after solid portion carry out extraction obtain leaching liquor, remove leaching liquor in dichloro
Methane and methanol obtain Nosiheptide crude extract, the Nosiheptide crude extract is washed to obtain Nosiheptide with n-hexane refined
Thing.
Wherein, in the mixing digestion agent of dichloromethane and methanol, the volume ratio of dichloromethane and methanol is 0.3-7:1.
In the case of preferable, in order to further improve the extracting effect of Nosiheptide, the volume ratio of dichloromethane and methanol is
1-6:1。
Wherein, the solid portion after the Nosiheptide production fermentation is by the fermentation to being obtained after Nosiheptide production fermentation
Product carries out what separation of solid and liquid was obtained.Had no particular limits in the present invention for carrying out the method for separation of solid and liquid, as long as energy
The enough moisture relatively sufficiently excluded in tunning, can use this area routine carries out solid-liquid point to tunning
From mode, such as can using stand, centrifugation, by the way of plate-frame filtering.Preferably, the row of extruding by way of plate-frame filtering
Go out moisture, expansion drying is carried out after obtaining wet filter cake.Obtain drying crude product, moisture is about 5%~8%.It will dry thick
Product are crushed by pulverizer, finally give the solid portion after the i.e. described Nosiheptide production fermentation of nosipeptide crude product powder.
The present invention has no particular limits for carrying out the specific species of the bacterial strain of Nosiheptide production fermentation, can be ability
Domain is known or unknown any bacterial strain that can carry out Nosiheptide fermenting and producing.In a kind of preferred embodiment of the present invention
In, the tunning is to ferment to be obtained in Nosiheptide fermentation medium using actinomyces streptomyces actuosus
The tunning obtained.
The present invention has no particular limits for the component of Nosiheptide fermentation medium, as long as can be used in training by fermenting
Support Nosiheptide production bacterial strain and obtain the tunning containing Nosiheptide by fermentation process well known in the art.
Preferably, the step of extraction is also included the mixing digestion agent and the solid part after Nosiheptide fermenting and producing first
Tap touches mixing, obtains acid soil, ultrasonic mixing is carried out to the acid soil, specifically, can be by the acid soil
Ultrasound mixes 0.5-1h under the ultrasonic wave of 50-150 hertz.
Preferably, ultrasound mix after by the acid soil at 38-42 DEG C extracting at constant temperature 0.8-1.2h.
Further, in one embodiment of the invention, in order to obtain preferable extracting effect, preferably soaked in constant temperature
Acid soil is transferred to oscillator under 150-200rpm after carrying and shakes 2-4h.
In order to sufficiently realize the extraction of Nosiheptide, leach step twice is preferably carried out, after first time extracts, from
Precipitation is separately recovered in the heart and leaching liquor, the precipitation of recovery are extracted with the mixing digestion agent of new dichloromethane and methanol again,
Step and condition extract identical with first time.
Optionally, relative to the solid portion after the production fermentation of 100g Nosiheptides, the mixing of the dichloromethane and methanol
The dosage of digestion agent is 600-1000ml.
When being extracted twice, extraction for the first time and second of volume ratio for extracting mixing digestion agent used are 2-3：
1。
Optionally, the dichloromethane in vacuum distillation removal leaching liquor is carried out to the leaching liquor and methanol obtains Nosiheptide
Crude extract.
In a kind of preferred embodiment of the present invention, using the mode of vacuum distillation by the dichloromethane in leaching liquor
Removed with methanol, vacuum distillation is preferably carried out under 40-70 DEG C of water bath condition.
In the present invention, relative to 100g Nosiheptide crude extracts, the dosage of n-hexane used in washing operation each time
For 80-150ml.
Preferably, washing step includes cleaning Nosiheptide crude extract with n-hexane, is centrifuged after cleaning and obtains solid
Crude extract is precipitated, then solid crude extract precipitation is cleaned with water.The step can be carried out 1-3 times.
Specific washing step can include：Carried out sufficiently after the Nosiheptide crude extract is contacted into mixing with n-hexane
Concussion washing, centrifuged after concussion washing and obtain solid crude extract precipitation.The solid crude extract is precipitated again and contacted with pure water
Centrifugation removes supernatant after carrying out fully shaking washing.Repeat the above steps until centrifuged supernatant is that colourless transparent liquid is
Material after only being washed.Wherein, precipitated relative to the every 100g solid crude extract, water used in each round cleaning
Dosage is 80-150ml.
In the case of particularly preferably, in order to further improve the purity of Nosiheptide, methods described, which is additionally included in, uses pure water
Concussion washing and centrifugation at least once are carried out after cleaning to material after the washing with n-hexane, until centrifuged supernatant is nothing
Untill color transparency liquid.
In method provided by the present invention, the tunning can utilize any of Nosiheptide life in this area
Production bacterial strain and fermentation process carry out the tunning obtained after Nosiheptide production fermentation.
Present invention also offers purposes of the method for the present invention in feed addictive is prepared.
Using method provided by the present invention, technical process present in existing Nosiheptide extraction process can be overcome numerous
It is miscellaneous, production cycle length, the defects of production process disposal of pollutants is more.The efficiency of production process is improved, reduces the wasting of resources and environment
Pollution, obtain the Nosiheptide product that purity is not less than 96.2%.
Culture presevation information：BJX004, Classification And Nomenclature are S.actuosus Streptomyces actuosus, and preservation is compiled
Number it is CGMCC No.8732, China Committee for Culture Collection of Microorganisms's commonly micro- life was preserved on 01 17th, 2014
Thing center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode：
100101。
Embodiment
With reference to embodiment, the present invention is described in detail.
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Unless otherwise specified, embodiment
In the conventional meanses that are well known to those skilled in the art of technological means used, raw materials used is commercial goods.
In following examples, the process of Nosiheptide production fermentation is：
1st, inclined-plane culture：
By S.actuosus BJX004 (Classification And Nomenclatures：S.actuosus Streptomyces actuosus, deposit number：
CGMCC No.8732, it was preserved on 01 17th, 2014 in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart) it is inoculated on slant medium, cultivated 7-8 days under the conditions of being 50-60% in 27.5 DEG C, envionmental humidity.
2nd, seed culture
Take the inclined-plane lawn 1cm obtained in step 12, accessed the kind equipped with seed culture medium sterilized 30ml
Sub- bottle, cultivate 24 hours under the following conditions：Temperature is 28 DEG C, envionmental humidity 50-60%, rotating speed 180-
200rpm, radius of turn 50mm, obtain first order seed nutrient solution.
First order seed nutrient solution is inoculated in secondary seed tank by 10% (V/V) inoculum concentration, cultivated under the following conditions
48 hours：Temperature is 28 DEG C, envionmental humidity 50-60%, rotating speed 180-200rpm, radius of turn 50mm, is obtained
Secondary seed nutrient solution.
3rd, fermented and cultured
Fermentation culture method：Take above-mentioned secondary seed nutrient solution to be inoculated in by 10% (V/V) inoculum concentration to go out equipped with 30ml
Cross in the triangular flask of the fermentation medium of bacterium, under the following conditions fermented and cultured 200 hours：Temperature is 28 DEG C, humidity 60%,
Rotating speed is 230rpm, radius of turn 50mm, obtains zymotic fluid, puts Nosiheptide unit 2500u/ml in tank zymotic fluid.
4th, after the completion of fermenting, by plate-frame filtering, abundant extruding discharge moisture obtains that to carry out flash distillation after wet filter cake dry
It is dry, 180 DEG C of EAT, 60~70 DEG C of leaving air temp.Obtain drying crude product, moisture is 5%~8%.Will be dried
Crude product is crushed by pulverizer, finally gives nosipeptide crude product powder, i.e. solid portion after Nosiheptide fermenting and producing.
Embodiment 1
50g nosipeptide crude product powder is taken, uses the mixing digestion agent of the dichloromethane and methanol (body of dichloromethane and methanol
Product is than being 2：1) 500ml is extracted.Extraction suspension is put into 100 hertz of mixing 1h of ultrasound in Ultrasound Instrument first, is put into after taking-up
1h is extracted in 40 DEG C of waters bath with thermostatic control, is transferred to oscillator 150rpm concussions 4h.
Concussion pours into system in 100ml centrifuge tubes after terminating, and 4000rpm centrifugation 10min, filtering, obtains supernatant.Return
Precipitation is received, all precipitations are mixed again, use mixing digestion agent (dichloromethane and the first of 200ml dichloromethane and methanol
The volume ratio of alcohol is 2：1) carry out second to extract, the same previous step of leach extraction method.
The clarification leaching liquor after all filterings is concentrated, is transferred in distilling flask, 40-70 DEG C of water-bath is evaporated under reduced pressure, and extraction is molten
After agent is evaporated, Nosiheptide crude extract is obtained.
100ml n-hexanes are added into distilling flask, the Nosiheptide crude extract that will attach on distilling flask is washed down, and is turned
Move on in 100ml centrifuge tube, cleaned using 80ml n-hexanes fully shaking, 4000rpm centrifugation 10min, obtain solids crude and carry
Thing precipitates.
Pure water 50ml is added into centrifuge tube, fully shaking cleaning, 4000rpm centrifugation 10min, then removes supernatant
Liquid, cleaned again with pure water, untill centrifuged supernatant is without color；
Precipitation after n-hexane 50ml is cleaned with upper step mixes, fully shaking cleaning precipitation, 4000rpm centrifugation 10min,
Remove supernatant；
The step of step is washed with n-hexane in repetition, untill centrifuged supernatant is colourless transparent liquid；Obtain that
Western peptide refines thing.
The Nosiheptide after cleaning is dried at dark drying and refines thing precipitation, is put into 50 DEG C of insulating box and dries after drying
4h, is put into constant weight in drier, and dried object is pulverized into powder, is transferred in dry brown bottle, and it is to be checked to deposit in -20 DEG C of sealings；
Weigh the refined things of about 0.004g to be put into 100ml brown volumetric flasks, 10ml phosphoric acid is added into brown volumetric flask
Salt buffer, 80ml DMF, is jiggled, and is put into ultrasonic 30min in Ultrasound Instrument, refined thing fully dissolved, mixed；
After mixing, solution is settled to 100ml with DMF；
Take 4ml or so solution to carry out millipore filter filtering, filtrate is filtered in brown sample injection bottle；
Sample introduction needle is cleaned, solution is extracted and cleans 2-3 times, finally extract 20 μ l sample liquids, and ensure bubble-free.Open sample introduction
Mouthful, sample introduction is then shut off injection port, starts to detect (Detection wavelength 241nm, flow velocity 1ml/min, 35 DEG C of column temperature, mobile phase=second
Nitrile：0.025% phosphate=1:1st, retention time 8-9min)
Highly finished product are calculated in standard curve by the sample of gained and peak area, sample weighting amount, the extension rate of standard specimen
Content.
The essence for obtaining drying powder processed carries nosiheptide powder 2.6g, and the purity that Nosiheptide extract is detected through liquid phase is 96.2%,
Yield is 94.3%.
Embodiment 2
Nosipeptide crude product powder is purified using method same as Example 1, differed only in, dichloromethane and
In the mixing digestion agent of methanol, the volume ratio of dichloromethane and methanol is 1:1.
The essence for obtaining drying powder processed carries nosiheptide powder 2.5g, and the purity that Nosiheptide extract is detected through liquid phase is 92.1%,
Yield is 91%.
Embodiment 3
Nosipeptide crude product powder is purified using method same as Example 1, differed only in, dichloromethane and
In the mixing digestion agent of methanol, the volume ratio of dichloromethane and methanol is 7:1.
The essence for obtaining drying powder processed carries nosiheptide powder 2.15g, and the purity that Nosiheptide extract is detected through liquid phase is 93.2%,
Yield is 89%.
Comparative example 1
Nosipeptide crude product powder is purified using method same as Example 1, differed only in, by dichloromethane
The dichloromethane of equivalent is replaced with the mixing digestion agent of methanol.
The essence for obtaining drying powder processed carries nosiheptide powder 2.12g, and the purity that Nosiheptide extract is detected through liquid phase is
87.35%, yield 90.2%.
Comparative example 2
Nosipeptide crude product powder is purified using method same as Example 1, differed only in, by dichloromethane
The methanol of equivalent is replaced with the mixing digestion agent of methanol.
The essence for obtaining drying powder processed carries nosiheptide powder 2.4g, and the purity that Nosiheptide extract is detected through liquid phase is 85.42%,
Yield is 88.9%.
Comparative example 3
Nosipeptide crude product powder is purified using method same as Example 1, differed only in, dichloromethane and
In the mixing digestion agent of methanol, the volume ratio of dichloromethane and methanol is 8:1.
The essence for obtaining drying powder processed carries nosiheptide powder 1.98g, and the purity that Nosiheptide extract is detected through liquid phase is 87.5%,
Yield is 85.8%.
Comparative example 4
Nosipeptide crude product powder is purified using method same as Example 1, differed only in, the temperature of extraction
For 44 DEG C.
The essence for obtaining drying powder processed carries nosiheptide powder 2.1g, and the purity that Nosiheptide extract is detected through liquid phase is 81.4%,
Yield is 88%.
Comparative example 5
Nosipeptide crude product powder is purified using method same as Example 1, differed only in, mixes digestion agent
Dosage be 1200ml.
The essence for obtaining drying powder processed carries nosiheptide powder 1.45g, and the purity that Nosiheptide extract is detected through liquid phase is 80%, is received
Rate is 78%.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a kind of Nosiheptide method of purification, it is characterised in that using the mixing digestion agent of dichloromethane and methanol to that west
Solid portion after peptide production fermentation carries out extraction and obtains leaching liquor, removes the dichloromethane in leaching liquor and methanol obtains that west
Peptide crude extract, the Nosiheptide crude extract is washed with n-hexane to obtain the refined thing of Nosiheptide.
2. according to the method for claim 1, it is characterised in that in the mixing digestion agent of dichloromethane and methanol, dichloro
The volume ratio of methane and methanol is 0.3-7:1.
3. method according to claim 1 or 2, it is characterised in that the condition of extraction includes, and is extracted at 38-42 DEG C
0.8-1.2h。
4. according to the method for claim 3, it is characterised in that relative to the solid part after the production fermentation of 100g Nosiheptides
Point, the dosage of the mixing digestion agent of the dichloromethane and methanol is 600-1000ml.
5. the method according to claim 1 or 4, it is characterised in that carry out being evaporated under reduced pressure removal extraction to the leaching liquor
Dichloromethane and methanol in liquid obtain Nosiheptide crude extract.
6. according to the method for claim 5, it is characterised in that relative to 100g Nosiheptide crude extracts, the n-hexane
Dosage is 80-150ml.
7. the method according to claim 1 or 6, it is characterised in that washing step includes slightly carrying Nosiheptide with n-hexane
Thing is cleaned, and centrifugation obtains solid crude extract precipitation after cleaning, then solid crude extract precipitation is cleaned with water.
8. the method according to claim 1 or 6, it is characterised in that methods described is included to being obtained after Nosiheptide production fermentation
The tunning obtained carries out separation of solid and liquid, obtains the solid portion after Nosiheptide production fermentation.
9. according to the method for claim 8, it is characterised in that the tunning is to utilize actinomyces streptomyces
Actuosus ferments obtained tunning in Nosiheptide fermentation medium.
10. purposes of the method in claim 1-9 described in any one in feed addictive is prepared.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109796520A (en) * | 2019-02-20 | 2019-05-24 | 山东齐发药业有限公司 | A kind of extracting method of Nosiheptide fine work |
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| CN101302247A (en) * | 2008-06-24 | 2008-11-12 | 浙江汇能动物药品有限公司 | Method for extracting nosipeptide crude product |
| CN101624418A (en) * | 2009-08-03 | 2010-01-13 | 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 |
| CN105198958A (en) * | 2015-09-29 | 2015-12-30 | 浙江汇能动物药品有限公司 | Nosiheptide finemeal extracting method |
| CN105420319A (en) * | 2015-12-24 | 2016-03-23 | 山东胜利生物工程有限公司 | Preparation method of pure nosiheptide |
-
2016
- 2016-07-15 CN CN201610560825.8A patent/CN107619429A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101302247A (en) * | 2008-06-24 | 2008-11-12 | 浙江汇能动物药品有限公司 | Method for extracting nosipeptide crude product |
| CN101624418A (en) * | 2009-08-03 | 2010-01-13 | 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 |
| CN105198958A (en) * | 2015-09-29 | 2015-12-30 | 浙江汇能动物药品有限公司 | Nosiheptide finemeal extracting method |
| CN105420319A (en) * | 2015-12-24 | 2016-03-23 | 山东胜利生物工程有限公司 | Preparation method of pure nosiheptide |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109796520A (en) * | 2019-02-20 | 2019-05-24 | 山东齐发药业有限公司 | A kind of extracting method of Nosiheptide fine work |
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