CN106977595A - The extracting method of one boar derived antimicrobial peptide - Google Patents
The extracting method of one boar derived antimicrobial peptide Download PDFInfo
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- CN106977595A CN106977595A CN201710321969.2A CN201710321969A CN106977595A CN 106977595 A CN106977595 A CN 106977595A CN 201710321969 A CN201710321969 A CN 201710321969A CN 106977595 A CN106977595 A CN 106977595A
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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Abstract
The invention discloses the extracting method of a boar derived antimicrobial peptide, the chitterlings after pre-treatment are heat-treated 15 25s in 80 100 DEG C of water first, clasmatosis more fully purpose when reaching raising raw material utilization rate and multigelation, then chitterlings homogenate is prepared, multigelation 56 times after homogenate and water for injection are well mixed according to mass ratio 100: 90 99, then by homogenate and acetic acid（Analysis is pure）It is well mixed according to mass ratio 100: 1 10, boiling water bath, cooling centrifugation, collects sediment and a supernatant, then sediment is well mixed with 2.87% acetic acid solution according to mass ratio 1: 0.8 1 respectively, refrigerated centrifuge, secondary supernatant fluid is collected, supernatant sequentially passes through tangential flow filtration, ultrafiltration after merging, regulation pH value is simultaneously filtered again, finally gives pig derived antimicrobial peptide.The inventive method can improve the utilization rate of raw material, and loss of the reduction tissue mashing process to instrument, while making clasmatosis during multigelation more abundant, improves the recovery rate of pig derived antimicrobial peptide.
Description
Technical field
The invention belongs to technical field of pharmaceutical biotechnology, and in particular to the extracting method of a boar derived antimicrobial peptide.
Background technology
Antibacterial peptide is a kind of active peptides with strong antibacterial action that organism produces, and is the important of biological congenital immunity
Constituent.So far, a variety of endogenous antibacterial activities are found in many biologies are including animal, plant and prokaryotes
Peptide.Pig derived antimicrobial peptide is isolated antibacterial peptide out of pig body.
Because antibacterial peptide has the advantages that broad-spectrum bactericidal action, relative molecular weight be smaller, thermally-stabilised and good water solubility, more
It is important that antibacterial peptide may act on prokaryotic and the eukaryotic for occurring lesion and be difficult to make pathogenic microorganism produce resistance
The characteristics of；Therefore, under the Tough questions for the global drug resistance that current mankind abuse of antibiotics is brought, for understanding in pig body
The function of spontaneous antibacterial peptide, and design a kind of method to prepare pig derived antimicrobial peptide in the hope of the research of substitute antibiotics,
Just seem particularly important, this certainly will will play positive facilitation to the research and application of finding Substitutes For Antibiotic.
Prior art is during slightly pig derived antimicrobial peptide tissue mashing is carried, when being smashed to pieces because containing larger tissue block, being homogenized,
Machine can be caused compared with macrolesion, and raw material utilization rate can be reduced；When tissue mashing is not abundant enough, multigelation can be influenceed
When clasmatosis degree, the extraction of final influence antibacterial peptide.
The content of the invention
It is an object of the invention to provide a kind of utilization rate for improving raw material, damage of the reduction tissue mashing process to instrument
Consumption, while making the extracting method of clasmatosis more fully pig derived antimicrobial peptide during multigelation.
To achieve the above object, the present invention is adopted the following technical scheme that：
The extracting method of one boar derived antimicrobial peptide, comprises the following steps：
1) chitterlings that are fresh or thawing are taken, manadesma, adipose tissue and the small intestine contents on chitterlings surface are removed, with injection
Rinsed well with water, chitterlings be then heat-treated 15-25s in 80-100 DEG C of water, rubbed after rapid cooling with meat grinder,
Then it is homogenized 3 times with colloid mill, obtains chitterlings homogenate；
2）Above-mentioned homogenate is well mixed with water for injection according to mass ratio 100: 90-99, it is even after being then well mixed
Slurries, which are sub-packed in apyrogenic container, to be placed in freezing in jelly storehouse, completely rear taking-up to be frozen, then is placed in solution in room temperature flowing water
Freeze, such multigelation 5-6 times；
3）By the homogenate and acetic acid after multigelation（Analysis is pure）It is well mixed according to mass ratio 100: 1-10, in 4 DEG C of stirrings
Mixed liquor is obtained after 12 hours, by mixed liquor boiling water bath 8-15 min, then mixed liquor is placed in frozen water and cooled down rapidly, from
The heart, collects sediment and a supernatant respectively, by a supernatant in 4 DEG C of preservations；
4）Above-mentioned sediment is well mixed with the acetic acid solution of concentration 2.87% according to mass ratio 1: 0.8-1, it is small in 4 DEG C of stirrings 12
When obtain leaching liquor, by leaching liquor below -10 DEG C of temperature, freezing 2-4 hours after（There is ice slag in liquid top layer to be leached, stops
Freezing）Centrifugation, collects secondary supernatant fluid；
5）Merge a supernatant and secondary supernatant, the supernatant after merging is sequentially passed through into 0.22 μm -0.45 μm and 5
KD-10 kD filter membranes carry out tangential flow filtration, collect filter membrane permeate, obtain ultrafiltrate；Ultrafiltrate pH value is adjusted to 3.0-6.5
Between, then through 0.1 μm of -0.22 μm of bacterial filter filtering, obtain pig derived antimicrobial peptide.
Further, the step 2）, the mass ratio of homogenate and water for injection is 100: 97.13.
Further, the step 3）, the mass ratio of homogenate and acetic acid after multigelation is 100: 2.87.
Further, the step 3）, mixed liquor carries out boiling water bath under agitation.
Further, the step 3）, the mixed liquor after cooling is in 4 DEG C, 6000-7500 r/min centrifugation 20-30 min.
Further, the step 4）, the mass ratio of sediment and 2.87% acetic acid solution is 1: 1.
Further, the step 4）, the leaching liquor after freezing is in 4 DEG C, 6000-7500 r/min centrifugation 20-30 min.
The present invention uses above technical scheme, before chitterlings cooling is rubbed, increases a heat treatment step, i.e., at early stage
Small intestine after reason handles 15-25s, then rapid cooling in temperature is 80-100 DEG C of hot water, then shreds, smashs to pieces, this method can
During so that chitterlings being homogenized in meat grinder and colloid mill, the ratio that chitterlings are milled is nonheat-treated thinner, hardly has big
The tissue of particle is present, and not only improves the utilization rate of raw material, and can reduce the damage to instrument.Simultaneously as being not present
The tissue block of bulky grain, the multigelation process then carried out, can more fully smudge cells, discharge more pig sources and resist
Bacterium peptide.
Step 3 of the present invention）, mixed liquor is during boiling water bath, and mixer should be stirred continuously heated equal to ensure
It is even), the homogenate after multigelation is well mixed with acetic acid according to mass ratio 100: 1-10, preferably 1: 2.87%, 2.87%
The difference that acetic acid solution extraction polypeptides matter content is not only extracted with 5% acetic acid solution is not obvious, and 2.87% acetic acid is molten
The antibacterial peptide solution osmotic pressure that liquid is extracted is the half that 5% acetic acid solution is extracted, it is to avoid the too high phenomenon of osmotic pressure occur.
Embodiment
The extracting method of one boar derived antimicrobial peptide, comprises the following steps：
1) chitterlings that are fresh or thawing are taken, manadesma, adipose tissue and the small intestine contents on chitterlings surface is removed, is rushed with water
Then chitterlings are heat-treated 15-25s by wash clean in 80-100 DEG C of water, are rubbed, are then used with meat grinder after rapid cooling
Colloid mill is homogenized 3 times, obtains chitterlings homogenate；
2）Above-mentioned homogenate is well mixed with water for injection according to mass ratio 100: 90-99, it is even after being then well mixed
Slurries, which are dispensed into apyrogenic container, to be placed in freezing in jelly storehouse, completely rear taking-up to be frozen, then is placed in solution in room temperature flowing water
Freeze, such multigelation 5-6 times；
3）By the homogenate and acetic acid after multigelation（Analysis is pure）It is well mixed according to mass ratio 100: 1-10, in 4 DEG C of stirrings
Mixed liquor is obtained after 12 hours, then mixed liquor is placed in fast in frozen water by mixed liquor boiling water bath 8-15min under agitation
Quickly cooling but, in 4 DEG C, 6000-7500 r/min centrifugation 20-30 min, collects sediment and a supernatant respectively, will once on
Clear liquid is in 4 DEG C of preservations；
4）Above-mentioned sediment is well mixed with the acetic acid solution of concentration 2.87% according to mass ratio 1: 0.8-1, it is small in 4 DEG C of stirrings 12
When after obtain leaching liquor, by leaching liquor below -10 DEG C of temperature, freezing 2-4 hours after（There is ice slag in liquid top layer to be leached, stops
Only freeze）In 4 DEG C, 6000-7500 r/min centrifugation 20-30 min, secondary supernatant fluid is collected；
5）Merge a supernatant and secondary supernatant, the supernatant after merging is sequentially passed through into 0.22 μm -0.45 μm and 5
KD -10 kD filter membranes carry out tangential flow filtration, collect filter membrane permeate, obtain ultrafiltrate；Ultrafiltrate pH value is adjusted to 3.0-7.0
Between, then through 0.1 μm of -0.22 μm of bacterial filter filtering, obtain pig derived antimicrobial peptide.
Embodiment 1
The extracting method of one boar derived antimicrobial peptide, comprises the following steps：
1) chitterlings that are fresh or thawing are taken, manadesma, adipose tissue and the small intestine contents on chitterlings surface is removed, is rushed with water
Then chitterlings are heat-treated 20s by wash clean in 90 DEG C of water, are rubbed after rapid cooling with meat grinder, are then used colloid mill
Homogenate 3 times, obtains chitterlings homogenate；
2）Above-mentioned homogenate is well mixed with water for injection according to mass ratio 100: 97.13, it is even after being then well mixed
Slurries, which are sub-packed in apyrogenic container, to be placed in freezing in jelly storehouse, completely rear taking-up to be frozen, then is placed in solution in room temperature flowing water
Freeze, such multigelation 6 times；
3）By the homogenate and acetic acid after multigelation（Analysis is pure）It is well mixed according to mass ratio 100: 2.87, in 4 DEG C of stirrings
Mixed liquor is obtained after 12 hours, then mixed liquor is placed in fast in frozen water by the mixed liquor min of boiling water bath 10 under agitation
Quickly cooling but, centrifuges 25 min in 4 DEG C, 7000 r/min, sediment and a supernatant is collected respectively, by a supernatant in 4
DEG C preserve；
4）Above-mentioned sediment is well mixed with the acetic acid solution of concentration 2.87% according to mass ratio 1: 1, after 4 DEG C are stirred 12 hours
Leaching liquor is obtained, by leaching liquor at -10 DEG C of temperature, after freezing 3 hours（There is ice slag in liquid top layer to be leached, stops freezing）In
4 DEG C, 7000 r/min, 25 min of centrifugation, collect secondary supernatant fluid；
5）Merge a supernatant and secondary supernatant, the supernatant after merging is sequentially passed through 0.22 μm and 10 kD filter membranes enter
Row tangential flow filtration, collects filter membrane permeate, obtains ultrafiltrate；Ultrafiltrate pH value is adjusted to 4.5, then through 0.1 μm of bacterial filter
Filtering, obtains pig derived antimicrobial peptide.
Embodiment 2
The extracting method of one boar derived antimicrobial peptide, comprises the following steps：
1) chitterlings that are fresh or thawing are taken, manadesma, adipose tissue and the small intestine contents on chitterlings surface is removed, is rushed with water
Then chitterlings are heat-treated 25s by wash clean in 80 DEG C of water, are rubbed after rapid cooling with meat grinder, are then used colloid mill
Homogenate 3 times, obtains chitterlings homogenate；
2）Above-mentioned homogenate is well mixed with water for injection according to mass ratio 100: 90, the homogenate after being then well mixed
It is sub-packed in apyrogenic container and is placed in freezing in jelly storehouse, completely rear taking-up to be frozen, then defrosting in room temperature flowing water is placed in, such as
This multigelation 6 times；
3）By the homogenate and acetic acid after multigelation（Analysis is pure）It is well mixed according to mass ratio 100: 10, stir 12 in 4 DEG C
Mixed liquor is obtained after hour, then mixed liquor is placed in frozen water rapid cold by mixed liquor boiling water bath 8min under agitation
But, 30 min are centrifuged in 4 DEG C, 6000 r/min, sediment and a supernatant is collected respectively, by a supernatant in 4 DEG C of guarantors
Deposit；
4）Above-mentioned sediment is well mixed with the acetic acid solution of concentration 2.87% according to mass ratio 1: 0.8, stirred 12 hours in 4 DEG C
Leaching liquor is obtained, by leaching liquor under the conditions of -24 DEG C, after freezing 2 hours（There is ice slag in liquid top layer to be leached, stops freezing）In
4 DEG C, 6000r/min 30 min of centrifugation, collect secondary supernatant fluid；
5）Merge a supernatant and secondary supernatant, the supernatant after merging is sequentially passed through 0.45 μm and 5kD filter membranes are carried out
Tangential flow filtration, collects filter membrane permeate, obtains ultrafiltrate；Ultrafiltrate pH value is adjusted to 3.0, then through 0.1 μm of bacterial filter mistake
Filter, obtains pig derived antimicrobial peptide.
Embodiment 3
The extracting method of one boar derived antimicrobial peptide, comprises the following steps：
1) chitterlings that are fresh or thawing are taken, manadesma, adipose tissue and the small intestine contents on chitterlings surface is removed, is rushed with water
Then chitterlings are heat-treated 15s by wash clean in 100 DEG C of water, are rubbed after rapid cooling with meat grinder, are then used colloid mill
Homogenate 3 times, obtains chitterlings homogenate；
2）Above-mentioned homogenate is well mixed with water for injection according to mass ratio 100: 99, the homogenate after being then well mixed
It is sub-packed in apyrogenic container and is placed in freezing in jelly storehouse, completely rear taking-up to be frozen, then defrosting in room temperature flowing water is placed in, such as
This multigelation 5 times；
3）By the homogenate and acetic acid after multigelation（Analysis is pure）It is well mixed according to mass ratio 100: 1, stir 12 in 4 DEG C
Mixed liquor is obtained after hour, then mixed liquor is placed in frozen water rapidly by the mixed liquor min of boiling water bath 15 under agitation
Cooling, centrifuges 20 min in 4 DEG C, 7500 r/min, sediment and a supernatant is collected respectively, by a supernatant in 4 DEG C
Preserve；
4）Above-mentioned sediment is well mixed with the acetic acid solution of concentration 2.87% according to mass ratio 1: 1, after 4 DEG C are stirred 12 hours
Leaching liquor is obtained, by leaching liquor at -10 DEG C of temperature, after freezing 4 hours（There is ice slag in liquid top layer to be leached, stops freezing）In
4 DEG C, 7500 r/min centrifugation 20min, collect secondary supernatant fluid；
5）Merge a supernatant and secondary supernatant, the supernatant after merging is sequentially passed through 0.3 μm and 7kD filter membranes are carried out
Tangential flow filtration, collects filter membrane permeate, obtains ultrafiltrate；Ultrafiltrate pH value is adjusted to 7.0, then through 0.22 μm of bacterial filter mistake
Filter, obtains pig derived antimicrobial peptide.
Claims (7)
1. the extracting method of a boar derived antimicrobial peptide, it is characterised in that：It comprises the following steps：
1) chitterlings are taken, manadesma, adipose tissue and the small intestine contents on chitterlings surface is removed, is rinsed well with water, then will
Chitterlings are heat-treated 15-25s in 80-100 DEG C of water, are rubbed after cooling with meat grinder, are then homogenized with colloid mill, obtain pig
Small intestine homogenate liquid；
2）Above-mentioned homogenate is well mixed with water for injection according to mass ratio 100: 90-99, it is even after being then well mixed
Slurries multigelation 5-6 times；
3）Homogenate after multigelation is well mixed with acetic acid according to mass ratio 100: 1-10, after 4 DEG C are stirred 12 hours
Mixed liquor is obtained, by mixed liquor boiling water bath 8-15min, then mixed liquor is placed in frozen water and cooled down, precipitation is collected in centrifugation respectively
Thing and a supernatant, by a supernatant in 4 DEG C of preservations；
4）Above-mentioned sediment is well mixed with 2.87% acetic acid solution according to mass ratio 1: 0.8-1, after 4 DEG C are stirred 12 hours
Leaching liquor is obtained, by leaching liquor below -10 DEG C of temperature, freezing is centrifuged after 2-4 hours, collects secondary supernatant fluid；
5）Merge a supernatant and secondary supernatant, the supernatant after merging is sequentially passed through into 0.22 μm -0.45 μm and 5
KD-10 kD filter membranes carry out tangential flow filtration, collect filter membrane permeate, obtain ultrafiltrate；Ultrafiltrate pH value is adjusted to 3.0-7.0
Between, then through 0.1 μm of -0.22 μm of bacterial filter filtering, obtain pig derived antimicrobial peptide.
2. the extracting method of boar derived antimicrobial peptide according to claim 1, it is characterised in that：The step 2）, homogenate
The mass ratio of liquid and water for injection is 100: 97.13.
3. the extracting method of boar derived antimicrobial peptide according to claim 1, it is characterised in that：The step 3）, repeatedly
The mass ratio of homogenate and acetic acid after freeze thawing is 100: 2.87.
4. the extracting method of boar derived antimicrobial peptide according to claim 1, it is characterised in that：The step 3）, mixing
Liquid carries out boiling water bath under agitation.
5. the extracting method of boar derived antimicrobial peptide according to claim 1, it is characterised in that：The step 3）, cooling
Mixed liquor afterwards is in 4 DEG C, 6000-7500 r/min centrifugation 20-30 min.
6. the extracting method of boar derived antimicrobial peptide according to claim 1, it is characterised in that：The step 4）, precipitation
The mass ratio of thing and 2.87% acetic acid solution is 1: 1.
7. the extracting method of boar derived antimicrobial peptide according to claim 1, it is characterised in that：The step 4）, freezing
Leaching liquor afterwards is in 4 DEG C, 6000-7500 r/min centrifugation 20-30 min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107345207A (en) * | 2017-06-29 | 2017-11-14 | 王德亮 | With animal intestinal mucosa production enteric microorganism powder co-production small-molecular peptides, liquaemin, the method for alkaline phosphatase and application |
| CN108939044A (en) * | 2018-09-30 | 2018-12-07 | 派生特（福州）生物科技有限公司 | A kind of preparation method of chicken intestinal peptide injection |
| CN109464652A (en) * | 2018-11-28 | 2019-03-15 | 派生特（福州）生物科技有限公司 | A kind of composition and its preparation method and application for poultry diarrhea |
| CN111729068A (en) * | 2020-07-06 | 2020-10-02 | 派生特（福州）生物科技有限公司 | Pet external spray and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103417574A (en) * | 2013-08-08 | 2013-12-04 | 福州派生特生物科技有限公司 | Preparation method of recombinant antimicrobial peptide extracted from small intestine of pig and application thereof |
-
2017
- 2017-05-09 CN CN201710321969.2A patent/CN106977595A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103417574A (en) * | 2013-08-08 | 2013-12-04 | 福州派生特生物科技有限公司 | Preparation method of recombinant antimicrobial peptide extracted from small intestine of pig and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107345207A (en) * | 2017-06-29 | 2017-11-14 | 王德亮 | With animal intestinal mucosa production enteric microorganism powder co-production small-molecular peptides, liquaemin, the method for alkaline phosphatase and application |
| CN108939044A (en) * | 2018-09-30 | 2018-12-07 | 派生特（福州）生物科技有限公司 | A kind of preparation method of chicken intestinal peptide injection |
| CN109464652A (en) * | 2018-11-28 | 2019-03-15 | 派生特（福州）生物科技有限公司 | A kind of composition and its preparation method and application for poultry diarrhea |
| CN109464652B (en) * | 2018-11-28 | 2022-02-01 | 派生特（福州）生物科技有限公司 | Composition for livestock diarrhea and preparation method and application thereof |
| CN111729068A (en) * | 2020-07-06 | 2020-10-02 | 派生特（福州）生物科技有限公司 | Pet external spray and preparation method thereof |
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