CN104000851A - Snake venom extract capable of replacing antibiotic and preparation method and application thereof - Google Patents
Snake venom extract capable of replacing antibiotic and preparation method and application thereof Download PDFInfo
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- CN104000851A CN104000851A CN201410212366.5A CN201410212366A CN104000851A CN 104000851 A CN104000851 A CN 104000851A CN 201410212366 A CN201410212366 A CN 201410212366A CN 104000851 A CN104000851 A CN 104000851A
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Abstract
The invention belongs to the technical field of agricultural biology and discloses a snake venom extract capable of replacing antibiotic and a preparation method and an application thereof. The preparation method comprises the following steps: (1) after dissolving the snake venom by a buffered saline solution, centrifuging to remove undissolved substances, adsorbing the obtained supernate by cation exchange resin and eluting the adsorption part by the corresponding buffered saline solution containing 1mol/L sodium chloride to obtain a snake venom component eluate; (2) ultra-filtering the snake venom component eluate by an ultrafiltration membrane for intercepting the components with the relative molecular mass less than 5-20 thousand and nano-filtering by a nanofiltration membrane to remove salt so as to obtain a snake venom compound extracting liquid; (3) freeze-drying the obtained snake venom compound extracting liquid to freeze-dried powder which is the snake venom extract capable of replacing antibiotic. The snake venom extract provided by the invention is capable of replacing antibiotic and is used for preventing and treating bacterial infectious poultry and livestock diseases such as staphylococcus aureus, escherichia coli and salmonella.
Description
Technical field
The invention belongs to agricultural biological technical field, be specifically related to a kind of alternative antibiotic snake venom extract and preparation method thereof and application.
Background technology
The annual nearly 6000 tons of antibiotic of China are for animal feeding.A large amount of antibiotic uses have not only caused the degeneration of Immune Function In Animals, and have caused drug resistance generally to occur.Some antibiotic remainss in animal body, to human body have produced adverse consequences after being eaten by people; Some antibiotic are discharged in environment from animal, cause environmental pollution, and the mankind's safety is constituted a threat to.
In antibiotic substitute, microbial fermentation preparation, Chinese herbal and crude drugs preparations, functional oligosaccharide, organic acid are the focuses of studying at present.Though life-time service has the effect of certain enhancing Immune Function In Animals, but its antibacterial effect is still poor compared with antibiotic, and the cost of life-time service is higher.
Snake venom is the powdery solid that the venom of Serpentis secretion forms through lyophilizing, and its main component is protease and polypeptide.Though snake venom is noxious substance, under doses, be safe.Clinically can treat tumor, digestive tract ulcer, thrombosis etc. with snake venom.Li Yunlong etc. reported multiple snake venom have certain antibacterial action (Li Yunlong, Zhang Hong. the antibacterial action of snake venom. Anhui Chinese Medicine College journal, 1995,14 (1): 54-55), show that snake venom contains antimicrobial component.
Snake venom antimicrobial component, through extracting, to reduce snake venom toxicity, and is improved to its antibacterial effect, be developed into the control of safe antibiotic substitute for animal livestock and poultry.Research has no report in this respect.
Summary of the invention
In order to overcome the shortcoming and deficiency of prior art, primary and foremost purpose of the present invention is to provide a kind of preparation method of alternative antibiotic snake venom extract;
Another object of the present invention is to provide the antibiotic snake venom extract that replaces that above-mentioned preparation method obtains;
A further object of the present invention is to provide the above-mentioned application that replaces antibiotic snake venom extract.
Object of the present invention is achieved through the following technical solutions:
A preparation method for alternative antibiotic snake venom extract, comprises the following steps:
(1) after snake venom is dissolved with buffered saline solution, the centrifugal insoluble matter of removing, gained supernatant cationic exchange resin adsorption, then with the corresponding buffered saline solution eluting absorbed portion containing 1mol/L sodium chloride, obtain snake venom component eluent;
Preferably, described snake venom is agkistrodon halyx pallas venom and cobra venom;
Preferably, described buffered saline solution is that pH is 3～6 phosphate or acetate buffer;
Preferably, described centrifugal condition is turn/min of rotating speed 3000～10000, centrifugation time 10～60min;
Preferably, described cation exchange resin is styrene storng-acid cation exchange resin, styrene weak-acid cation-exchange resin or acrylic acid weak-acid cation-exchange resin;
Preferably, 1～5 times of the quality that the consumption of described cation exchange resin is described snake venom;
Preferably, the adsorption time of described supernatant in cation exchange resin is 2～12h;
Preferably, 2～5 times of the quality that the consumption of the described corresponding buffered saline solution containing 1mol/L sodium chloride is described cation exchange resin;
(2) step (1) gained snake venom component eluent is carried out to ultrafiltration by ultrafilter membrane, hold back the component that relative molecular mass is less than 0.5～20,000, and carry out nanofiltration with except freshen by NF membrane, obtain snake venom component extracting solution;
Preferably, the pressure of described ultrafiltration is 0.1～0.5MPa;
Preferably, the pressure of described nanofiltration is 0.5～2MPa;
(3) described snake venom component extracting solution is carried out to lyophilization and become lyophilized powder, obtain described alternative antibiotic snake venom extract.
A kind of alternative antibiotic snake venom extract being obtained by above-mentioned preparation method.
Above-mentioned alternative antibiotic snake venom extract in prevention and treatment bacterial infection livestock and poultry as the application of antibiotic substitute;
Preferably, described alternative antibiotic snake venom extract in prevention and treatment staphylococcus aureus, escherichia coli or Salmonella infection livestock and poultry as the application of antibiotic substitute.
Principle of the present invention:
The present invention through cationic exchange resin adsorption, has removed snake venom most of acid fibrinolysin, then has passed through ultrafilter membrane ultrafiltration, removes neurotoxin; Use nanofiltration desalination, and lyophilization, the extract toxicity obtaining significantly reduces, and has retained antibacterial activity component.
The present invention has following advantage and effect with respect to prior art:
(1) preparation method of the present invention has been removed the strong macromole enzyme of ophidism, has reduced the toxicity of described alternative antibiotic snake venom extract to animal, but has retained antibacterial activity, and its safety and activity are significantly improved.
(2) preparation method of the present invention does not need, by each snake venom component process column chromatography for separation, to be applicable to suitability for industrialized production, and cost is lower.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
A kind of alternative antibiotic snake venom extract, its preparation method is as follows:
(1) get agkistrodon halyx pallas venom 10g, the sodium-acetate buffer that is 3 with the pH of 100mL dissolves, the centrifugal 10min of 10000 turn/min, gained supernatant adsorbs 12h with 50g acrylic acid weak-acid cation-exchange resin, the sodium-acetate buffer eluting resin containing 1mol/L sodium chloride with 100g, obtains snake venom component eluent again;
(2) gained snake venom component eluent is carried out to ultrafiltration by the ultrafilter membrane of 20,000 molecular weight, ultrafiltration pressure is 0.1MPa, carries out nanofiltration to remove freshen again by ultrafilter membrane part through NF membrane, and nanofiltration pressure 2.0MPa, obtains snake venom component extracting solution;
(3) by the lyophilization of gained snake venom component extracting solution, obtain lyophilized powder 2.8g, obtain described alternative antibiotic snake venom extract.
Embodiment 2
A kind of alternative antibiotic snake venom extract, its preparation method is as follows:
(1) get agkistrodon halyx pallas venom 10g, the sodium phosphate buffer that is 6 with the pH of 100mL dissolves, the centrifugal 60min of 3000 turn/min, gained supernatant adsorbs 2h with 10g styrene storng-acid cation exchange resin, the sodium phosphate buffer eluting resin containing 1mol/L sodium chloride with 50g, obtains snake venom component eluent again;
(2) gained snake venom component eluent is carried out to ultrafiltration by the ultrafilter membrane of 0.5 ten thousand molecular weight, ultrafiltration pressure 0.5MPa, carries out nanofiltration to remove freshen through NF membrane again by ultrafilter membrane part, and nanofiltration pressure 0.5MPa, obtains snake venom component extracting solution;
(3) by the lyophilization of gained snake venom component extracting solution, obtain lyophilized powder 1.1g, obtain described alternative antibiotic snake venom extract.
Embodiment 3
(1) get agkistrodon halyx pallas venom 10g, the sodium-acetate buffer that is 5 with the pH of 100mL dissolves, the centrifugal 30min of 5000 turn/min, gained supernatant adsorbs 6h with 30g styrene weak-acid cation-exchange resin, the sodium-acetate buffer eluting resin containing 1mol/L sodium chloride with 100g, obtains snake venom component eluent again;
(2) gained snake venom component eluent is carried out to ultrafiltration by the ultrafilter membrane of 10,000 molecular weight, ultrafiltration pressure 0.3MPa, carries out nanofiltration to remove freshen through NF membrane again by ultrafilter membrane part, and nanofiltration pressure 1.0MPa, obtains snake venom component extracting solution;
(3) by the lyophilization of gained snake venom component extracting solution, obtain lyophilized powder 2.4g, obtain described alternative antibiotic snake venom extract.
Embodiment 4
A kind of alternative antibiotic snake venom extract, its preparation method is as follows:
(1) get cobra venom 10g, the sodium phosphate buffer that is 3 with the pH of 100mL dissolves, the centrifugal 20min of 6000 turn/min, gained supernatant adsorbs 12h with 50g styrene weak-acid cation-exchange resin, the sodium phosphate buffer eluting resin containing 1mol/L sodium chloride with 150g, obtains snake venom component eluent again;
(2) gained snake venom component eluent is carried out to ultrafiltration by the ultrafilter membrane of 20,000 molecular weight, ultrafiltration pressure 0.2MPa, carries out nanofiltration to remove freshen through NF membrane again by ultrafilter membrane part, and nanofiltration pressure 2MPa, obtains snake venom component extracting solution;
(3) by the lyophilization of gained snake venom component extracting solution, obtain lyophilized powder 3.6g, obtain described alternative antibiotic snake venom extract.
Embodiment 5
A kind of alternative antibiotic snake venom extract, its preparation method is as follows:
(1) get cobra venom 10g, the sodium-acetate buffer that is 4 with the pH of 100mL dissolves, the centrifugal 50min of 3000 turn/min, gained supernatant adsorbs 10h with 30g acrylic acid weak-acid cation-exchange resin, 50g, containing the sodium-acetate buffer eluting resin of 1mol/L sodium chloride, obtains snake venom component eluent again;
(2) gained snake venom component eluent is carried out to ultrafiltration by the ultrafilter membrane of 10,000 molecular weight, ultrafiltration pressure 0.4MPa, carries out nanofiltration to remove freshen through NF membrane again by ultrafilter membrane part, and nanofiltration pressure 1.0MPa, obtains snake venom component extracting solution;
(3) by the lyophilization of gained snake venom component extracting solution, obtain lyophilized powder 1.9g, obtain described alternative antibiotic snake venom extract.
Embodiment 6
A kind of alternative antibiotic snake venom extract, its preparation method is as follows:
(1) get cobra venom 10g, the sodium-acetate buffer that is 3 with the pH of 100mL dissolves, the centrifugal 10min of 10000 turn/min, gained supernatant adsorbs 5h with 20g styrene storng-acid cation exchange resin, the sodium-acetate buffer eluting resin containing 1mol/L sodium chloride with 100g, obtains snake venom component eluent again;
(2) gained snake venom component eluent is carried out to ultrafiltration by the ultrafilter membrane of 0.5 ten thousand molecular weight, ultrafiltration pressure 0.5MPa, carries out nanofiltration to remove freshen through NF membrane again by ultrafilter membrane part, and nanofiltration pressure 0.5MPa, obtains snake venom component extracting solution;
(3) by the lyophilization of gained snake venom component extracting solution, obtain lyophilized powder 1.4g, obtain described alternative antibiotic snake venom extract.
Embodiment 7
The alternative antibiotic snake venom extract of getting embodiment 1～6 preparation carries out the antibiotic property experiment to poultry:
Method: the experimental bacteria liquid of isolated escherichia coli, staphylococcus aureus, Salmonella from diarrhoea pig and sick chicken secretions, join respectively in MH broth bouillon, be mixed with 10
8the concentration of cfu/mL, in 37 DEG C of cultivation 6～8h, for subsequent use.
Adopt in vitro medicinal liquid dilution method, the normal saline solution 2.0mL of described alternative antibiotic snake venom extract (concentration is 50 μ g/mL) is doubly to dilution with meat soup, puts into 37 DEG C of incubators together with after mixing with above-mentioned 3 kinds of test organisms liquid 0.1mL respectively to cultivate 16～24h.By the meat soup transferred species of the limpid antibacterial that do not grow of a pipe minimum described alternative antibiotic snake venom extract concentration, to agar plate, still without bacterial growth, the dose that represents this pipe is exactly the minimal inhibitory concentration (MIC) of theasapogenol derivant.Adopt gentamycin as positive control drug.
Result: the testing result of the minimum inhibitory concentration of described alternative antibiotic snake venom extract to pathogenic bacterium is as shown in table 1, analyzed from table 1, the alternative antibiotic snake venom extract of embodiment 1～6 preparation all has good inhibitory action to three kinds of malignant bacterias, its minimum inhibitory concentration is suitable with positive control drug gentamycin, illustrates that alternative antibiotic snake venom extract of the present invention has antibiotic therapeutic effect.
The minimum inhibitory concentration (1 μ g/mL) of table 1 alternative antibiotic snake venom extract of the present invention to pathogenic bacterium
Embodiment 8
The alternative antibiotic snake venom extract that embodiment 1～6 is made, joins in the drinking water of antibacterial infection poultry, and per kilogram dosage is 0.01g, and without animal dead, livestock and poultry control is good.Show that described alternative antibiotic snake venom extract toxicity is low, good effect.
Embodiment 9
The alternative antibiotic snake venom extract that embodiment 1～6 is made, with after physiological saline solution, gives antibacterial and infects livestock and poultry muscle injection, and dosage is 0.1mg/kg/d, and for three days on end, without animal dead, disease control status is good.Show that described alternative antibiotic snake venom extract toxicity is low, good effect.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (6)
1. a preparation method for alternative antibiotic snake venom extract, is characterized in that comprising the following steps:
(1) after snake venom is dissolved with buffered saline solution, the centrifugal insoluble matter of removing, gained supernatant cationic exchange resin adsorption, then with the corresponding buffered saline solution eluting absorbed portion containing 1mol/L sodium chloride, obtain snake venom component eluent;
(2) step (1) gained snake venom component eluent is carried out to ultrafiltration by ultrafilter membrane, hold back the component that relative molecular mass is less than 0.5～20,000, and carry out nanofiltration with except freshen by NF membrane, obtain snake venom component extracting solution;
(3) described snake venom component extracting solution is carried out to lyophilization and become lyophilized powder, obtain described alternative antibiotic snake venom extract.
2. the preparation method of a kind of alternative antibiotic snake venom extract according to claim 1, is characterized in that: in step (1):
Described snake venom is agkistrodon halyx pallas venom and cobra venom;
Described buffered saline solution is that pH is 3～6 phosphate or acetate buffer;
Described centrifugal condition is turn/min of rotating speed 3000～10000, centrifugation time 10～60min;
The consumption of described cation exchange resin is 1～5 times of quality of described snake venom;
Described cation exchange resin is styrene storng-acid cation exchange resin, styrene weak-acid cation-exchange resin or acrylic acid weak-acid cation-exchange resin;
2～5 times of the quality that the consumption of the described corresponding buffered saline solution containing 1mol/L sodium chloride is described cation exchange resin.
3. the preparation method of a kind of alternative antibiotic snake venom extract according to claim 1, is characterized in that: in step (2): the pressure of described ultrafiltration is 0.1～0.5MPa; The pressure of described nanofiltration is 0.5～2MPa.
4. the alternative antibiotic snake venom extract obtaining according to the preparation method described in claim 1～3 any one.
Alternative antibiotic snake venom extract according to claim 4 in prevention and treatment bacterial infection livestock and poultry as the application of antibiotic substitute.
6. application according to claim 5, is characterized in that: described alternative antibiotic snake venom extract in prevention and treatment staphylococcus aureus, escherichia coli or Salmonella infection livestock and poultry as the application of antibiotic substitute.
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| CN201410212366.5A CN104000851B (en) | 2014-05-19 | 2014-05-19 | A kind of snake venom extract of alternative antibiotic and preparation method and application |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108776222A (en) * | 2018-06-14 | 2018-11-09 | 吉林省农业科学院 | Gongzhuling mycin bacteriostatic activity detection method and application |
| CN109929020A (en) * | 2017-12-15 | 2019-06-25 | 浙江京新药业股份有限公司 | A kind of purification process of cobra venom and products thereof |
Citations (1)
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| CN101265296A (en) * | 2008-04-08 | 2008-09-17 | 中国科学院昆明动物研究所 | Reptile cathelicidin antibiotic peptide and derivatives, and application thereof |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101265296A (en) * | 2008-04-08 | 2008-09-17 | 中国科学院昆明动物研究所 | Reptile cathelicidin antibiotic peptide and derivatives, and application thereof |
Non-Patent Citations (4)
| Title |
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| CHELLAPANDI P等: "Purification and Antibacterial Activity of Indian Cobra and Viper Venoms", 《ELECTRONIC JOURNAL OF BIOLOGY》 * |
| 张白羽等: "蛇毒抗菌肽OH-CATH对大肠杆菌引起家兔泌尿系感染的保护作用", 《动物学研究》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109929020A (en) * | 2017-12-15 | 2019-06-25 | 浙江京新药业股份有限公司 | A kind of purification process of cobra venom and products thereof |
| CN108776222A (en) * | 2018-06-14 | 2018-11-09 | 吉林省农业科学院 | Gongzhuling mycin bacteriostatic activity detection method and application |
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