CN102174067A - Method for producing MD insect antibacterial peptide intermediate - Google Patents
Method for producing MD insect antibacterial peptide intermediate Download PDFInfo
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- CN102174067A CN102174067A CN2011100323659A CN201110032365A CN102174067A CN 102174067 A CN102174067 A CN 102174067A CN 2011100323659 A CN2011100323659 A CN 2011100323659A CN 201110032365 A CN201110032365 A CN 201110032365A CN 102174067 A CN102174067 A CN 102174067A
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Abstract
The invention provides a method for producing a musca domestica (MD) insect antibacterial peptide intermediate, and aims to reduce waste in a production process, simplify the production process and reduce production cost to ensure that the MD insect antibacterial peptide intermediate can be industrially produced. In order to achieve the effects, an MD antibacterial peptide is extracted by a small molecular peptide, the used reagent is deionized water, and ethanol is removed from a mobile phase A and a mobile phase B by controlling the temperature and vacuum degree through vacuum distilled water by hydrophobic chromatography. Due to the technical scheme, the process is simplified, the production cost is reduced, a high extraction ratio still can be achieved by a unique hydrophobic chromatography process without protein electrophoresis or a process of recycling the small molecular peptide through electrophoresis, cost is reduced, the production rate is improved, and a product can be popularized.
Description
Technical field
The present invention relates to a kind of production method of antibacterial peptide, relate in particular to a kind of production method of MD insect antimicrobial peptide intermediate.
Background technology
Insect is a biological group of occurring in nature maximum, and the mankind make discovery from observation, and all is distributed with various insects in what kind of ecotope.Can for where how insect survive under the rugged environment? through research, it is found that insect after being upset or infecting, in its hemolymph, can produce a kind of anti-microbial type material, claim antibacterial peptide.Antibacterial peptide has that molecule is little, good stability, broad-spectrum antimicrobial, characteristics such as have no side effect, and is the important factor that insect can be resisted the invasion and attack of nature harmful microorganism.And the most effective with the MD insect antimicrobial peptide in the insect antimicrobial peptide, the MD insect is the abbreviation of housefly Musca domestica, gets generic name and first letter of planting name in the Latin formal name used at school.Housefly has the close habit that contacts with pathogenic micro-organism, but to pathogenic micro-organism very strong resistibility should be arranged.Shown in " the Musca domestica larva antimicrobial substance is formed and physico-chemical property " literary composition that scientists such as Wang Yuancheng are delivered in 1997 " microorganism journal " and found that there is antimicrobial substance in housefly, and the MD insect antimicrobial peptide has wide practical use in fields such as agricultural, medicine, food.
Again there is the people that house fly antibiotic peptide is separated thereafter.Disclose in volume the 5th phase " comparisons of two kinds of extracting method of the insect antimicrobial peptide " literary composition " Chinese media biology and control magazine " October the 17th in 2006 adopts acupuncture infection escherichia coli to induce the generation antibacterial peptide to Musca domestica larva, extract its antibacterial peptide, and handle with physiological saline or two kinds of methods of phosphoric acid buffer through grinding, chromatography by cells were tested by flow cytometry.
In addition, present domesticly induce separating technology several below also having according to what applicant retrieval was recognized to the MD insect antimicrobial peptide:
As seen, the extraction for the MD insect antimicrobial peptide in the prior art has suitable proven technique, but shortcoming but is the production cost height, and craft precision requires high.The extraction of most MD insect antimicrobial peptide all is to finish in the laboratory, can't carry out industrial industrial large-scale production.So also caused the on the high side of MD insect antimicrobial peptide, can't be committed to actual clinical and use, can't bring benefit to the mankind.And in the production process of MD insect antimicrobial peptide, except final product, many intermediate products all are wasted, and have caused very big loss, have relatively improved production cost.
Summary of the invention
Technical problem to be solved by this invention provides a kind of production method of MD insect antimicrobial peptide intermediate, reduces the waste in the production technique, simplifies production technique, reduces production costs, and makes physical efficiency suitability for industrialized production in the middle of the MD insect antimicrobial peptide.
For solving the problems of the technologies described above, the present invention adopts following technical scheme: MD insect antimicrobial peptide intermediate production method is characterized in that: may further comprise the steps:
(1) the MD insect is made MD insect dry powder;
(2) will extract MD insect active albumen intermediate through the resulting MD insect of (1) step dry powder;
(3) will isolate MD insect small-molecule biologically active peptides intermediate through the resulting MD insect active of (2) step albumen intermediate;
(4) to being dissolved in the deionized water through the resulting MD small molecule active of step (3) peptide intermediate;
(5) solution that generates in (4) step is added in on the hydrophobic gradient and adsorption resin column of ether base as aglucon;
(6) hydrophobic gradient described in (5) step and adsorption resin column are regulated flow, by the binary gradient pump with different gradient eluent wash-outs;
(7) will be through the elutriant fraction collection of (6) step, ethanol is taken off in each component vacuum distilling;
(8) product in (7) step is delivered to Freeze Drying Equipment respectively freeze-drying obtain multiple MD insect antimicrobial peptide intermediate;
(9) waste water in each step is adopted follow-on microorganism absorption method handle.
Preferably, deionized water resistivity 〉=17.5 megaohms that adopt in described (4) step, the ratio of usage quantity is: 1000 gram small molecule active peptide intermediates use 5000-8000 gram deionized water.Select for use such deionized water and proportioning to make the middle physical efficiency of abundant MD small molecule active peptide fully be dissolved in the deionized water.
Preferably, the elutriant that adopts in described (6) step comprises mobile phase A and Mobile phase B, wherein the composition of mobile phase A is the ethanol of 2.5mol/L ammonium sulfate, 45mmol/L potassium primary phosphate and molar percentage 70%, and Mobile phase B is the ethanol of 45mmol/L potassium primary phosphate and molar percentage 50%.Carry the material that component moves forward and be called moving phase, be in equilibrium state with stationary phase.Concentration is determined concentration and hydrophobicity according to hydrophobicity in each moving phase, and hydrophobic is exactly to become estranged water molecules.Contain hydrophobic amino acid in protein and the polypeptide, these sour water acidic amino acids with hydrophobic group because of tending to cluster or agglomerating arrangement.Hydrophobic interaction chromatography be exactly according to the differential liberation of protein and peptide molecule surface hydrophobicity they.Elutriant is not to use a concentration always, but by raise gradually concentration or reduce concentration gradually of different moving phase, this effect realizes with the binary gradient pump.
Preferably, the flow velocity in described (6) step is 4mL/min-5.5mL/min.
Preferably, each component vacuum distilling is taken off the ethanol concrete steps and is in described (7) step: chuck stainless-steel vacuum distiller takes off ethanol, by controlling 80 ℃-85 ℃ and vacuum tightness 0.05Mpa---0.06Mpa collects ethanol, obtains taking off alcoholic acid liquid.
Preferably, described ethanol recycles.Can reduce production cost like this.
The present invention is owing to adopted technique scheme, simplified technology, reduced production cost, antibacterial peptide is one or more of MD insect small-molecule peptide, utilize small-molecular peptides to extract the MD antibacterial peptide, the reagent that uses is deionized water, and mobile phase A and Mobile phase B adopt hydrophobic chromatography to use vacuum distillation water management temperature and vacuum tightness to take off ethanol.The hydrophobic chromatography technology that employing is unique is not carried out protein electricity arteries and veins and electric arteries and veins recovery micromolecule polypeptide technology still can obtain high extraction yield, has reduced cost, has improved productivity, makes product to promote.
Embodiment
A kind of MD insect antimicrobial peptide of the present invention intermediate may further comprise the steps:
1.MD insect antimicrobial peptide intermediate production method is characterized in that: may further comprise the steps:
(1) the MD insect is made MD insect dry powder;
(2) will extract MD insect active albumen intermediate through the resulting MD insect of (1) step dry powder;
(3) will isolate MD insect small-molecule biologically active peptides intermediate through the resulting MD insect active of (2) step albumen intermediate;
2. (4) are to being dissolved in the deionized water through the resulting MD small molecule active of step (3) peptide intermediate; Deionized water resistivity 〉=17.5 megaohms that adopt in described (4) step, the ratio of usage quantity is: 1000 gram small molecule active peptide intermediates use 5000-8000 gram deionized water.
(5) solution that generates in (4) step is added in on the hydrophobic gradient and adsorption resin column of ether base as aglucon;
(6) hydrophobic gradient described in (5) step and adsorption resin column are regulated flow, by the binary gradient pump with different gradient eluent wash-outs; The elutriant that adopts in described (6) step comprises mobile phase A and Mobile phase B, wherein the composition of mobile phase A is the ethanol of 2.5mol/L ammonium sulfate, 45mmol/L potassium primary phosphate and molar percentage 70%, and Mobile phase B is the ethanol of 45mmol/L potassium primary phosphate and molar percentage 50%.Flow velocity is 4mL/min-5.5mL/min, and too fast or slow excessively flowing all can cause wash-out incomplete, reduces production efficiency.
(7) will be through the elutriant fraction collection of (6) step, ethanol is taken off in each component vacuum distilling; Each component vacuum distilling is taken off the ethanol concrete steps and is in the step: chuck stainless-steel vacuum distiller takes off ethanol, by controlling 80 ℃-85 ℃ and vacuum tightness 0.05Mpa---0.06Mpa collects ethanol, obtains taking off alcoholic acid liquid.Described ethanol recycles, reduces production costs, and energy-conserving and environment-protective.
(8) product in (7) step is delivered to Freeze Drying Equipment respectively freeze-drying obtain multiple MD insect antimicrobial peptide intermediate; Promptly can obtain the MD insect antimicrobial peptide intermediate of multiple different molecular weight, molecular weight mainly concentrates on 2000-8000.The antibacterial peptide of different molecular weight can destroy and kill different germ cell and cancer cells.
(9) waste water in each step is adopted follow-on microorganism absorption method handle, in MD insect protein intermediate production process, can produce waste water the present invention and design a new wastewater treatment step, comprise that waste water collected in each step compiles cooling at water collecting basin, water after the cooling in the water collecting basin is fed except that adopting biological adsorption and oxidation method to handle again after the slag bath slagging-off, the water collecting basin that goes back to again that biological adsorption and oxidation method is handled the back supernatant water recycles, all the other water outlets feed the tertiary treatment machine room and handle, and the water after the processing imports clean water basin and stores.Comprise coagulation apparatus, filter plant and ion-exchange unit in the described tertiary treatment machine room.Described biological adsorption and oxidation method comprises in the A section interconnective B section aeration tank and B section settling tank in the interconnective A section aeration tank and intermediate sedimentation pond and B section, microporous diffuser equipment and the artificial oxygen system of blast aeration device wherein are set in the A section aeration tank, and throw in Powdered Activated Carbon.A section aeration tank is moved with very high loads, and active sludge has certain adsorptive power.Throw in Powdered Activated Carbon, strengthen to the absorption of entering organic matter of water with to water inlet decolouring and microorganism organic matter degradation, and make stable embedding of gac play the effect that improves the active sludge precipitation threshold and suppress sludge bulking in the active sludge charcoal absorption.
Coagulation, filtration, ion exchange treatment can the degree of depth be removed residual microorganism, fine suspension, residual organic matter (being biodegradable organism mostly) in the water, blackout material and solvability salt.Water outlet after coagulation, filtration, the ion exchange treatment enters clean water basin, is transported to the production plant of MD insect antimicrobial peptide by the clean water basin water pump, as the production cycle water, and the conserve water resource.
What the present invention produced in process of production is the waste water of diversification, can not thoroughly clear up with simple method.Therefore the present invention improves biological adsorption and oxidation method of the prior art (AB method), make it more clear up more targetedly to the waste water of generation of the present invention, and the water quality that cleans out can apply to different occasions according to actual needs, meets very much environmental protection requirement.
The beneficial effect that invention is brought can show intuitively by following table:
As seen, be compared with existing technology the present invention and bring following advantage:
1, technology adopts from the activated protein intermediate of 1KgMD insect dry powder extraction about 55%, from the activated protein intermediate, extract 14% MD insect small-molecule peptide, extract 6.5% (5 gram) antibacterial peptide from small-molecular peptides, recovery rate is 0.50%, the recovery rate height;
2, according to MD insect antimicrobial peptide intermediate molecular size range, obtain multiple antibacterial peptide, can kill and wound and kill the Various Diseases bacterial cell;
3, only use deionized water in the technology, ammonium sulfate, potassium dihydrogen phosphate, ethanol and 5 kinds of main reagent of ether base, the original activity of MD insect active albumen intermediate and the special mechanism of absorption of MD insect small-molecule biologically active peptide intermediate there is not big impact, MD insect active albumen intermediate and MD insect small-molecule biologically active peptide intermediate still can be used for biological food, biological health-care product, the raw material midbody of biologics does not need again mutually to mend with the ion exchange layer analysis method simultaneously. Greatly reduce the production cost of MD insect antimicrobial peptide intermediate, only 5 dollars of the every gram production costs of the antibacterial peptide behind the purifying, 8 dollars/gram of selling price has so just been laid solid foundation for promoting; And product of the prior art all is higher than 10 dollars/gram.
4, technology is take MD insect small-molecule peptide intermediate as raw material, just formed MD insect active albumen intermediate, product (product) chain of MD insect small-molecule biologically active peptide intermediate and MD insect antimicrobial peptide intermediate and the MD insect essential oil of deriving, the products such as chitin, the senior organic fertilizer of fruits and vegetables, the biotic component of MD insect can obtain the full-scale development utilization;
5, technology makes the MD insect antimicrobial peptide intermediate can be low-cost, high added value large-scale production.
6, the output investment ratio produced of the product chain of MD small-molecular peptides, MD insect active albumen intermediate, MD insect antimicrobial peptide intermediate is bigger, if but three enterprises respectively go into operation a product, investment is just little, profit is considerable.
By end product MD insect antimicrobial peptide intermediate of the present invention be MD insect small-molecule peptide one or more, utilize small-molecular peptides to extract the MD antibacterial peptide, the reagent that uses is deionized water, and mobile phase A and Mobile phase B adopt hydrophobic chromatography to use vacuum distillation water management temperature and vacuum to take off ethanol. The hydrophobic chromatography technology that employing is unique is not carried out protein electricity arteries and veins and electric arteries and veins recovery micromolecule polypeptide technology still can obtain high recovery rate, has reduced cost, has improved productivity ratio, so that product can be promoted, is the technology of innovation.
Claims (6)
1.MD insect antimicrobial peptide intermediate production method is characterized in that: may further comprise the steps:
(1) the MD insect is made MD insect dry powder;
(2) will extract MD insect active albumen intermediate through the resulting MD insect of (1) step dry powder;
(3) will isolate MD insect small-molecule biologically active peptides intermediate through the resulting MD insect active of (2) step albumen intermediate;
(4) to being dissolved in the deionized water through the resulting MD small molecule active of step (3) peptide intermediate;
(5) solution that generates in (4) step is added in on the hydrophobic gradient and adsorption resin column of ether base as aglucon;
(6) hydrophobic gradient described in (5) step and adsorption resin column are regulated flow, by the binary gradient pump with different gradient eluent wash-outs;
(7) will be through the elutriant fraction collection of (6) step, ethanol is taken off in each component vacuum distilling;
(8) product in (7) step is delivered to Freeze Drying Equipment respectively freeze-drying obtain multiple MD insect antimicrobial peptide intermediate;
(9) waste water in each step is adopted follow-on microorganism absorption method handle.
2. according to the described MD insect antimicrobial peptide of claim 1 intermediate production method, it is characterized in that: deionized water resistivity 〉=17.5 megaohms that adopt in described (4) step, the ratio of usage quantity is: 1000 gram small molecule active peptide intermediates use 5000-8000 gram deionized water.
3. according to the described MD insect antimicrobial peptide of claim 1 intermediate production method, it is characterized in that: the elutriant that adopts in described (6) step comprises mobile phase A and Mobile phase B, wherein the composition of mobile phase A is the ethanol of 2.5mol/L ammonium sulfate, 45mmol/L potassium primary phosphate and molar percentage 70%, and Mobile phase B is the ethanol of 45mmol/L potassium primary phosphate and molar percentage 50%.
4. according to the described MD insect antimicrobial peptide of claim 1 intermediate production method, it is characterized in that: the flow velocity in described (6) step is 4mL/min-5.5mL/min.
5. according to the described MD insect antimicrobial peptide of claim 1 intermediate production method, it is characterized in that: each component vacuum distilling is taken off the ethanol concrete steps and is in described (7) step: chuck stainless-steel vacuum distiller takes off ethanol, by controlling 80 ℃-85 ℃ with vacuum tightness 0.05Mpa---0.06Mpa collects ethanol, obtains taking off alcoholic acid liquid.
6. according to the described MD insect antimicrobial peptide of claim 5 intermediate production method, it is characterized in that: described ethanol recycles.
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| CN2011100323659A CN102174067A (en) | 2011-01-28 | 2011-01-28 | Method for producing MD insect antibacterial peptide intermediate |
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| CN2011100323659A CN102174067A (en) | 2011-01-28 | 2011-01-28 | Method for producing MD insect antibacterial peptide intermediate |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392020A (en) * | 2008-11-06 | 2009-03-25 | 中山大学 | Musca domestic pupae natural antimicrobial peptide products and preparation method and use thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101392020A (en) * | 2008-11-06 | 2009-03-25 | 中山大学 | Musca domestic pupae natural antimicrobial peptide products and preparation method and use thereof |
Non-Patent Citations (6)
| Title |
|---|
| MOREIRA CK 等: "The Musca domestica larval hexamerin is composed of multiple, similar polypeptides", 《INSECT BIOCHEM MOL BIOL》 * |
| 刘绛光 等: "小分子肽分离方法研究进展", 《中国生化药物杂志》 * |
| 张爱君 等: "稀碱法提取食用蝇蛆蛋白", 《昆虫知识》 * |
| 张统 等: "生物吸附氧化法（AB法）的理论和实践", 《环境污染与防治》 * |
| 王芙蓉 等: "家蝇幼虫活性蛋白组分的提取及清除羟基自由基活性研究", 《昆虫知识》 * |
| 陆婕 等: "家蝇蛆抗菌肽提取工艺研究", 《昆虫学报》 * |
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Application publication date: 20110907 |