CN105154508A - Antimicrobial peptide preparation method - Google Patents
Antimicrobial peptide preparation method Download PDFInfo
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- CN105154508A CN105154508A CN201510620536.8A CN201510620536A CN105154508A CN 105154508 A CN105154508 A CN 105154508A CN 201510620536 A CN201510620536 A CN 201510620536A CN 105154508 A CN105154508 A CN 105154508A
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- antibacterial peptide
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Abstract
The invention discloses an antimicrobial peptide preparatoin method. The method includes the following steps that 1, cattle blood is taken, bromelain is added to crushed blood cells for enzymolysis; 2, acetic acid is added to enzymatic hydrolysate for extraction, supernatant is extracted in a centrifugation mode, the pH is adjusted to 6.0 +/-0.5, and cattle blood antibacterial peptide crude extracts are obtained; 3, the cattle blood antibacterial peptide crude extracts are purified; 4, freezing and drying are conducted. The preparation method is moderate in condition, free of pollution, high in efficiency and low in cost, provides a new way and method for producing antimicrobial peptide and can be applied to separation and purification of the antimicrobial peptide from fresh cattle blood on a large scale.
Description
Technical field
The invention belongs to biological technical field, particularly a kind of preparation method of antibacterial peptide.
Background technology
At present, along with the abuse of conventional antibiotic, bacterial resistance sex chromosome mosaicism and drug residue problem are on the rise, even there is superbacteria, and these problems also directly threaten the eubiosis in human health and even whole biosphere, cause the concern of the high degree in the world, and sought a kind of noresidue, novel antibacterial medicine of having no drug resistance replacing conventional antibiotic and become the task of top priority.Antibacterial peptide has efficient fungistatic effect and extensive and stable biologic activity, extensively causes interest and the concern of people.Antibacterial peptide not only has wide spectrum, efficiently antimicrobial acivity, and microorganism can not be made to produce resistance because of the Antibacterial Mechanism of its uniqueness, and noresidue in body, the development prospect that tool is wide, the application of antibacterial peptide will bring the revolution of a new antibiotic.
China, as cattle-raising big country of the world, ox blood abundance, develops insufficient, cheap, can meet the demand of extensive in-depth processing, have very strong Application and Development and promotional value.
Summary of the invention
The object of this invention is to provide a kind of preparation method of antibacterial peptide, its preparation method mild condition, pollution-free, efficiency is high.To achieve these goals, the present invention proposes following technical scheme:
A preparation method for antibacterial peptide, the method comprises the following steps:
(1) get ox blood, add bromeline after carrying out hemocyte fragmentation (object is release oxyphorase) and carry out enzymolysis;
(2) in enzymolysis solution, add Acetic Acid Extraction, centrifuging and taking supernatant, regulate pH to 6.0 ± 0.5 (as 6.0), obtain ox blood antibacterial peptide crude extract;
(3) described ox blood antibacterial peptide crude extract is splined on SephadexG-100 gel column, adopts elutriant to carry out wash-out, collect the elution peak with anti-microbial activity;
(4) elution peak with anti-microbial activity that step (3) is collected is splined on SephadexG-25 gel column, adopts elutriant to carry out wash-out, collect elution peak, obtain ox blood antibacterial peptide solution;
(5) the ox blood antiseptic solution lyophilize of step (4) being collected.
In the step (1) of described method, the proportioning of described bromeline and described ox blood can be 750-35000U:100ml (as 1750-3500U:100ml, concrete as 3500U:100mL); The temperature of described enzymolysis can be 70 ~ 90 DEG C (as 70 DEG C), and the time can be 4 ~ 6h (as 4h).In addition, in the present invention, carrying out hemocyte described in, broken especially by adopting tissue mashing machine to stir, described ox blood realizes.
In the present invention, the enzyme of described bromeline is lived as 3500U/mg.Accordingly, the proportioning of described bromeline and described ox blood can be 0.5-10mg:100ml (as 0.5-1mg:100ml, concrete as 1mg:100mL).
In the step (2) of described method, describedly in enzymolysis solution, add acetic acid specifically can be: the acetic acid aqueous solution adding isopyknic 5% (volumn concentration) in described enzymolysis solution; Described lixiviate specifically can be: 4 ~ 37 DEG C (as 4 DEG C ~ 10 DEG C, 4 DEG C for another example) concussion 16-20h (as 16h); Describedly centrifugally be: 6600g-10000g (as 10000g) 4 ~ 10 DEG C (as 4 DEG C) centrifugal 20min ~ 30min (as 20min).
In the step (3) of described method, described elutriant specifically can be: pH be 6.0 concentration be the aqueous sodium acetate solution of 0.2mol/L; The speed of described wash-out specifically can be 10.0mL/cm
2h.
In one embodiment of the invention, raw two elution peaks of common property in the elution process of step (3), described in have the elution peak of anti-microbial activity actual be second elution peak.Described anti-microbial activity is embodied as at least one in anti-following bacterium: intestinal bacteria (as ACTT25922), Pseudomonas aeruginosa (as ACTT27853), Aeromonas hydrophila (GenebankIDSequenceID:gb|KJ156374.1|).
In the step (4) of described method, described elutriant specifically can be: pH be 6.0 concentration be the aqueous sodium acetate solution of 0.2mol/L; The speed of described wash-out specifically can be 10.0mL/cm
2h.
In one embodiment of the invention, only produce an elution peak in the elution process of step (4), this elution peak is the ox blood antibacterial peptide sample after purifying.
In the step (3) of described method, the column length of SephadexG-100 gel column and column internal diameter are than being 8:1.Specifically, the SephadexG-100 gel column adopted in the present invention is Pharmacia Products, and its catalog number is 17-0061-02).The applied sample amount of described loading is 10ml; The elution volume of described wash-out is 1-1.5L.
In the step (4) of described method, the column length of SephadexG-25 gel column and column internal diameter are than being 8:1.Specifically, the SephadexG-25 gel column adopted in the present invention is Pharmacia Products, and its catalog number is 17-0033-1.The applied sample amount of described loading is 10ml; The elution volume of described wash-out is 1-1.5L.
In the above-mentioned methods, namely described ox blood can be fresh ox blood, also can be the ox blood melted after freezing.
Utilize method described above to extract the ox blood antibacterial peptide obtained and also belong to protection scope of the present invention.
Specifically, the present invention has the following advantages:
1, the inventive method achieves to extract from ox blood and has the peptide material of stronger anti-microbial activity, provides a new approaches and methods for producing antibacterial peptide.
2, the antibacterial peptide adopting the inventive method to extract, belongs to natural extract, has natural, nontoxic, pollution-free, noresidue, has no side effect, functional characteristics that high-efficiency antimicrobial, antiviral, growth promotion and immunity moderation etc. are extensive and stable.
3, compared with congenic method, the inventive method is easy to operate, and equipment is simple, and extracting cycle is short, and separation and Extraction rate is high.
4, the abundant raw material source of institute of the present invention development, production cost is low, and ox blood can be made to obtain higher value application.Therefore application of the present invention not only can bring good economic benefit, but also has good ecological benefits, and then also has important public hygienics meaning, meets the theory building economical and environment-friendly society.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1
1, fresh ox blood is collected from ox slaughterhouse, tissue mashing machine stirs (can be undertaken freezing for the ox blood of collection by upper step after the used time melts equally) again, the object of this step is broken hemocyte, and release oxyphorase, after freeze thawing, effect is better.
2, add bromeline in the ox blood processed to step 1, the proportioning of bromeline and ox blood is 1mg papoid/100ml ox blood stoste, and mixing, enzymolysis 4h under 70 DEG C of conditions, to obtain enzymolysis solution.
3, the enzymolysis solution to step 2 adds isopyknic 5% acetic acid (volumn concentration, solvent is water), 4 DEG C of concussion lixiviate 16h, 8000r/min (being equivalent to 10000g) 4 DEG C of centrifugal 20min, leave and take supernatant, adjust its pH value to 6.0, be the crude extract of ox blood antibacterial peptide.
4, (column length with internal diameter ratio is ox blood antibacterial peptide crude extract to be splined on SephadexG-100 gel column at 4 DEG C
8:1, SephadexG-100 gel column is Pharmacia Products, and its catalog number is 17-0061-02) carry out chromatography, applied sample amount is 10ml, elutriant to be pH be 6.0 concentration be the aqueous sodium acetate solution of 0.2mol/L, the speed of wash-out is 10.0mL/cm2h, and elution volume is 1L.Nucleic acid-protein detector detects, after automatic collector is collected, and registering instrument record.
There are 2 elution peaks in ox blood crude extract after SephadexG-100 gel filtration chromatography.The component of each for elution peak collection tube is got 20 μ L and carries out bacteriostatic test.Agarose diffusion method is adopted (to detect bacterium on agar plate
Final concentration be 106CFU/ml) to collect elutriant detect, detect and collect liquid to the anti-microbial effect of intestinal bacteria ACTT25922 strain, substitute antibacterial peptide as positive control using the mycillin solution of isopyknic 20000IU, and substitute antibacterial peptide as negative control using the sodium-acetate elutriant of isopyknic 0.2mol/L.Collect the elutriant with bacteriostatic action.
5, effective antibacterial peptide (the elutriant with bacteriostatic action of merging that step 4 is obtained, namely the 9-16 pipe collected after SephadexG-100 gel filtration chromatography) concentrated after use SephadexG-25 gel column further (column length and internal diameter be than being 8:1, the SephadexG-25 gel column adopted in the present invention is Pharmacia Products, its catalog number is 17-0033-1) carry out chromatography, applied sample amount is 10ml, elutriant to be pH be 6.0 concentration be the aqueous sodium acetate solution of 0.2mol/L, the speed of wash-out is 10.0mL/cm2h, elution volume is 1L.Nucleic acid-protein detector detects, after automatic collector is collected, and registering instrument record.Collect the elutriant with bacteriostatic action, preserve under being placed on-20 DEG C of conditions.
6, effective antibacterial peptide solution lyophilize step 5 obtained.
The antibacterial tests of the ox blood antibacterial peptide that embodiment 1 obtains
Adopt agarose disperse method, carry out anti-microbial activity detection to the ox blood antibacterial peptide that embodiment 1 is extracted from ox blood, measure antibacterial circle diameter size, concrete operations are with associated description in embodiment 1 step 4.Have for examination bacterium: intestinal bacteria ACTT25922, Pseudomonas aeruginosa ACTT27853 strain, Aeromonas hydrophila (GenebankIDSequenceID:gb|KJ156374.1|).Substitute antibacterial peptide with the mycillin solution of isopyknic 20000IU for each for examination bacterium all simultaneously, and substitute the negative control of antibacterial peptide with the sodium-acetate elutriant of isopyknic 0.2mol/L.
Test in triplicate, results averaged.
Result shows, the ox blood antibacterial peptide that embodiment 1 is extracted from ox blood all has obvious restraining effect to each strains tested, and negative control is all without anti-microbial activity.
Claims (5)
1. a preparation method for antibacterial peptide, comprises the steps:
(1) get ox blood, add bromeline after carrying out hemocyte fragmentation and carry out enzymolysis;
(2) in enzymolysis solution, add Acetic Acid Extraction, centrifuging and taking supernatant, regulate pH to 6.0 ± 0.5, obtain the crude extract of ox blood antibacterial peptide;
(3) crude extract of described ox blood antibacterial peptide is splined on SephadexG-100 gel column, adopts elutriant to carry out wash-out, collect the elution peak with anti-microbial activity;
(4) elution peak with anti-microbial activity that step (3) is collected is splined on SephadexG-25 gel column, adopts elutriant to carry out wash-out, collect elution peak, obtain described ox blood antibacterial peptide solution;
(5) the ox blood antibacterial peptide solution lyophilize of step (4) being collected.
2. the preparation method of a kind of antibacterial peptide according to claim 1, is characterized in that: in step (1), and the quality proportioning of described bromeline and described ox blood is 1750-35000U:100ml; And/or in step (1), the temperature of described enzymolysis is 70 ~ 90 DEG C, and the time is 4 ~ 6h.
3. according to the preparation method of a kind of antibacterial peptide according to claim 1, it is characterized in that: in step (2), the described acetic acid that adds in enzymolysis solution is: in described enzymolysis solution, add the acetic acid aqueous solution that isopyknic volumn concentration is 5%; And/or in step (2), described lixiviate is 4 ~ 37 DEG C of concussion 16-20h; And/or in step (2), described centrifugal be 6600 ~ 10000g4 DEG C of centrifugal 20 ~ 30min.
4., according to the preparation method of a kind of antibacterial peptide described according to claim 1, it is characterized in that: in step (3), described elutriant to be pH be 6.0 concentration be the aqueous sodium acetate solution of 0.2mol/L; And/or the speed of described wash-out is 10.0mL/cm2h.
5. according to the preparation method of a kind of antibacterial peptide described in claim 1, it is characterized in that: in step (4), described elutriant to be pH be 6.0 concentration be the aqueous sodium acetate solution of 0.2mol/L; And/or the speed of described wash-out is 10.0mL/cm
2h.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148463A (en) * | 2016-07-27 | 2016-11-23 | 柳州市柳南区安顺养殖协会 | A kind of preparation method of camel Sanguis Bovis seu Bubali antibacterial peptide |
| CN106148464A (en) * | 2016-07-27 | 2016-11-23 | 柳州市柳南区安顺养殖协会 | A kind of preparation method of Xinjiang brown Sanguis Bovis seu Bubali antibacterial peptide |
| CN107488224A (en) * | 2017-09-15 | 2017-12-19 | 吴光 | A kind of method that albumen is extracted with animal tissue |
| CN112521444A (en) * | 2020-12-16 | 2021-03-19 | 广东鑫皇冠新材料有限公司 | Extraction method of antibacterial peptide |
-
2015
- 2015-09-27 CN CN201510620536.8A patent/CN105154508A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106148463A (en) * | 2016-07-27 | 2016-11-23 | 柳州市柳南区安顺养殖协会 | A kind of preparation method of camel Sanguis Bovis seu Bubali antibacterial peptide |
| CN106148464A (en) * | 2016-07-27 | 2016-11-23 | 柳州市柳南区安顺养殖协会 | A kind of preparation method of Xinjiang brown Sanguis Bovis seu Bubali antibacterial peptide |
| CN107488224A (en) * | 2017-09-15 | 2017-12-19 | 吴光 | A kind of method that albumen is extracted with animal tissue |
| CN112521444A (en) * | 2020-12-16 | 2021-03-19 | 广东鑫皇冠新材料有限公司 | Extraction method of antibacterial peptide |
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Application publication date: 20151216 |