CN108048434A - The extracting method of earthworm protein and Lumbrokinase in a kind of earthworm - Google Patents
The extracting method of earthworm protein and Lumbrokinase in a kind of earthworm Download PDFInfo
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- CN108048434A CN108048434A CN201711462748.3A CN201711462748A CN108048434A CN 108048434 A CN108048434 A CN 108048434A CN 201711462748 A CN201711462748 A CN 201711462748A CN 108048434 A CN108048434 A CN 108048434A
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
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- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/303—Extraction; Separation; Purification by precipitation by salting out
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- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
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Abstract
The present invention relates to the extracting methods of earthworm protein and Lumbrokinase in a kind of earthworm, it is characterised in that using chromatographic column and the method for combining ultrafiltration, Lumbrokinase and earthworm protein are extracted from earthworm.This method realizes the sterile mechanism of protein extraction with radio-frequency technique, and the preference temperature that enzyme activity is controlled to occur, safety and sanitation are pollution-free, by realizing the extraction and separation of earthworm protein to the otherness control of earthworm protein, are conducive to the batch production of earthworm protein and Lumbrokinase.
Description
Technical field
The present invention relates to the extracting method of earthworm protein and Lumbrokinase in a kind of earthworm, more particularly to a kind of radio-frequency technique is auxiliary
The method for helping Lumbrokinase extraction and earthworm protein enzymolysis.
Background technology
Earthworm also known as earthworm, rich in functional components such as protein, amino acid, trace element, enzyme, hormones, the dry egg of earthworm
White matter content reaches 70%, is the raw material for preparing drug, cosmetics, health food etc., has very big economic value.Lumbrokinase
It is a kind of serine protein hydrolase with kinases and plasmin activity, can is as the drug for the treatment of thrombus disease
Two kind new medicines of the prevention and treatment cardiovascular and cerebrovascular disease of state approval are one of current most valuable fibrinolytic drugs.
Radio frequency (abbreviation RF) is a kind of high-frequency ac electromagnetic wave, and frequency range is 3kHz~300MHz.Early in 20th century,
Radio-frequency technique has just been applied to heat meat products, bread and production dehydrated vegetables external, the defrosting processing of frozen food with
The fields such as food sterilization, radio-frequency technique also early has application in China, but its application is only limitted to industry and medical treatment, should in field of food
With very few.Earthworm protein extraction efficiency is improved using the variation electromagnetic wave system of radio-frequency technique, stimulates earthworm because of the secretion of kinases,
So as to fulfill the industrialized production of earthworm kinases and short chain polypeptides.
The method of purification of Lumbrokinase is typically that single variety is saltoutd, and then passes through a variety of chromatographies and HPLC, FPLC or even parent
With chromatography or the means such as DEAE ion-exchange chromatographies, molecular sieve, gel electrophoresis, a variety of fibrinolytics are obtained from the earthworm of different lines
The simple component of enzyme, but it is complicated for operation, and long flow path, treating capacity is small, and enzyme activity loss is very big, and is not suitable for industrialized production.
It is a variety of active component compositions in view of Lumbrokinase, so in extraction using the work for being more suitable for industrialized production
Skill, molecule quantity space of the selection with biological activity protein are separated, and for the present invention for the purpose of industrialized production, selection is practical
Feasible process route, can specifically from earthworm homogenate one line obtain Lumbrokinase, superoxide dismutase, other
Enzyme, earthworm polypeptide, simple and practicable, extract active ingredient is high, is suitble to large-scale production.
The content of the invention
In view of this, the present invention is intended to provide a kind of earthworm protein and the method for Lumbrokinase extraction, this method have reaction
It is efficient, reaction condition is controllable, while the application of radio-frequency technique can be achieved sterilizing mechanism, enhancing enzyme activity enzymolysis rate.
In order to achieve the above objectives, the technical proposal of the invention is realized in this way：
The ancillary technique that the present invention is extracted using radio-frequency technique as Lumbrokinase and earthworm protein, extracts separation in earthworm
Earthworm protein, Lumbrokinase and enzymolysis polypeptide, specific implementation step are as follows：(1) fresh full earthworm living is taken, washed several times with water is most
Maximum possible discharges the dirt in its internal organ, then pulls out and inhales its excessive moisture as far as possible with filter paper；
(2) phosphate buffer for adding in the 0.1mol/LpH7.8 of 4 DEG C of precoolings is homogenized 5 minutes in homogenizer, in 4 DEG C of ice
When standing 4 is small in case, rear high speed centrifugation discards precipitation, obtains supernatant；
(3) taking supernatant reinforcing body ammonium sulfate, 4000r/min centrifuges 10min, collects supernatant up to 35% saturation degree, then
Solid ammonium sulfate is added in up to 70% saturation degree, is stood overnight, high speed centrifugation, is collected precipitation, use 0.1mol/L phosphate buffers afterwards
This sediment is dissolved, filters to obtain crude protein solution；
(4) crude protein solution is added into deionized water desalination, is concentrated into concentrate and is in neutrality (pH7.0), enrichment reaches 5%
When enter vacuum freeze drier drying 5-20 it is small when to get earthworm protein freeze-dried powder；
(5) phosphate buffer that earthworm protein freeze-dried powder is added in 0.1mol/LpH7.8 dissolves, and 20000da- is crossed at both ends
40000da ultrafiltration membranes obtain trapped fluid A and filtered solution B；
(6) trapped fluid A is added into deionized water desalination, is concentrated into concentrate and is in neutrality (pH7.0), when enrichment reaches 5%
Into vacuum freeze drier drying 5-20 it is small when, obtain lyophilized Lumbrokinase；
(7) crude protein solution obtained by step (3) is added in into protease hydrolyzed, is digested using RF assistance, crossed 6000da and surpass
Filter membrane by filtered solution plus deionized water desalination, is concentrated into concentrate and is in neutrality (pH7.0), enrichment enters vacuum when reaching 5%
When freeze drier drying 5-20 is small, 6500da ubiquitin-like earthworm polypeptides are obtained.
Phosphate buffer can also add in the sodium chloride of 0.1%-0.5% or sodium citrate or phosphoric acid in the step (2)
Disodium hydrogen or sodium acid carbonate or aqueous sodium carbonate.
Step (3) the high speed centrifugation is using 15000~25000r/min of rotating speed, horizontal spiral centrifuge.
Step (5) ultrafiltration membrane can also be bag filter, and dialyzate is 0.02mol/L phosphate buffers.
Chromatographic column filler is one kind in glucan or agarose in the step (5).
Protease refers to neutral proteinase and trypsase in the step (7).
The extracting method subsidiary conditions of earthworm protein and Lumbrokinase are the radio frequency of frequency 10MHz-200MHz in the earthworm
System.
Compared with the prior art, the extracting method of earthworm protein and Lumbrokinase in a kind of earthworm of the present invention has
Following advantage：
Low concentration sodium salt is added in phosphate buffer, using substep salting out method, saltout rate and protein yield can be improved.
Loss of proteins rate is high in ultrafiltration or dialysis, and protein molecule transmitance can be improved using RF assistance, reduces
Molecule loses and the reduction of enzyme activity.
The extraction process of Lumbrokinase and earthworm protein can realize the sterile mechanism of albumen, prevent protease microbiological contamination from going bad, product
Quality is more excellent.
Specific embodiment
With reference to embodiment, the present invention will be described in detail.
Embodiment 1
(1) fresh full earthworm living is taken, washed several times with water is as much as possible discharged the dirt in its internal organ, then pulled out
Its excessive moisture is inhaled as far as possible with filter paper, is placed in radio frequency system and handles 30min, rf frequency 100MHz, temperature is 37 DEG C；
(2) phosphate buffer of the 0.1mol/LpH7.8 of 4 DEG C of precoolings of addition and 0.2% sodium-chloride water solution are in homogenate
It is homogenized 5 minutes in device, it is rear to centrifuge when standing 4 is small in 4 DEG C of refrigerators, precipitation is discarded, obtains supernatant；
(3) taking supernatant reinforcing body ammonium sulfate, 4000r/min centrifuges 10min, collects supernatant up to 35% saturation degree, then
Solid ammonium sulfate is added in up to 70% saturation degree, is stood overnight, 15000r/min centrifugations collect precipitation, use 0.1mol/L phosphoric acid afterwards
This sediment of buffer solution filters to obtain crude protein solution.
(4) crude protein solution is added into deionized water desalination, is concentrated into concentrate and is in neutrality (pH7.0), enrichment reaches 5%
When enter vacuum freeze drier drying 10 it is small when to get earthworm protein freeze-dried powder.
Embodiment 2
(1) fresh full earthworm living is taken, washed several times with water is as much as possible discharged the dirt in its internal organ, then pulled out
Its excessive moisture is inhaled as far as possible with filter paper, is placed in radio frequency system and handles 30min, rf frequency 150MHz, temperature is 40 DEG C；
(2) phosphate buffer of the 0.1mol/LpH7.8 of 4 DEG C of precoolings of addition and 0.2% sodium citrate aqueous solution are in homogenate
It is homogenized 5 minutes in device, when standing 4 is small in 4 DEG C of refrigerators, rear high speed centrifugation discards precipitation, obtains supernatant；
(3) taking supernatant reinforcing body ammonium sulfate, 4000r/min centrifuges 10min, collects supernatant up to 35% saturation degree, then
Solid ammonium sulfate is added in up to 70% saturation degree, is stood overnight, 20000r/min centrifugations collect precipitation, use 0.1mol/L phosphoric acid afterwards
This sediment of buffer solution filters to obtain crude protein solution.
(4) crude protein solution is added into deionized water desalination, is concentrated into concentrate and is in neutrality (pH7.0), enrichment reaches 5%
When enter vacuum freeze drier drying 15 it is small when to get earthworm protein freeze-dried powder.
(5) earthworm protein freeze-dried powder is added in the phosphate buffer of the 0.1mol/LpH7.8 of 4 DEG C of precoolings, crosses glucan column,
20000da-40000da ultrafiltration membranes are crossed at both ends, obtain trapped fluid A and filtered solution B.
(6) trapped fluid A is added into deionized water desalination, is concentrated into concentrate and is in neutrality (pH7.0), when enrichment reaches 5%
Into vacuum freeze drier drying 15 it is small when, obtain lyophilized Lumbrokinase.
Embodiment 3
(1) fresh full earthworm living is taken, washed several times with water is as much as possible discharged the dirt in its internal organ, then pulled out
Its excessive moisture is inhaled as far as possible with filter paper, is placed in radio frequency system and handles 30min, rf frequency 200MHz, temperature is 50 DEG C；
(2) add in 4 DEG C of precoolings the phosphate buffer of 0.1mol/LpH7.8 and 0.2% disodium hydrogen phosphate aqueous solution in
It is homogenized 5 minutes in homogenizer, when standing 4 is small in 4 DEG C of refrigerators, rear high speed centrifugation discards precipitation, obtains supernatant；
(3) taking supernatant reinforcing body ammonium sulfate, 4000r/min centrifuges 10min, collects supernatant up to 35% saturation degree, then
Solid ammonium sulfate is added in up to 70% saturation degree, is stood overnight, 25000r/min centrifugations collect precipitation, use 0.1mol/L phosphoric acid afterwards
This sediment of buffer solution filters to obtain crude protein solution.
(4) crude protein solution is added in into neutral proteinase and trypsase, be placed in 150MHz radio frequency systems, cross 7000da
Bag filter obtains 6500da earthworm polypeptides.
Embodiment 4
(1) fresh full earthworm living is taken, washed several times with water is as much as possible discharged the dirt in its internal organ, then pulled out
Its excessive moisture is inhaled as far as possible with filter paper, is placed in radio frequency system and handles 30min, rf frequency 30MHz, temperature is 40 DEG C；
(2) phosphate buffer of the 0.1mol/LpH7.8 of 4 DEG C of precoolings of addition and 0.2% sodium citrate aqueous solution are in homogenate
It is homogenized 5 minutes in device, when standing 4 is small in 4 DEG C of refrigerators, rear high speed centrifugation discards precipitation, obtains supernatant；
(3) taking supernatant reinforcing body ammonium sulfate, 4000r/min centrifuges 10min, collects supernatant up to 35% saturation degree, then
Solid ammonium sulfate is added in up to 70% saturation degree, is stood overnight, 25000r/min centrifugations collect precipitation, use 0.1mol/L phosphoric acid afterwards
This sediment of buffer solution filters to obtain crude protein solution.
(4) crude protein solution is added into deionized water desalination, is concentrated into concentrate and is in neutrality (pH7.0), enrichment reaches 5%
When enter vacuum freeze drier drying 15 it is small when to get earthworm protein freeze-dried powder.
(5) earthworm protein freeze-dried powder is placed in 300MHz radio frequencies, adds in the phosphoric acid of the 0.1mol/LpH7.8 of 4 DEG C of precoolings
Buffer solution crosses agar column, and both ends cross 20000da-40000da ultrafiltration membranes, obtain trapped fluid A and filtered solution B.
(6) trapped fluid A is added into deionized water desalination, is concentrated into concentrate and is in neutrality (pH7.0), when enrichment reaches 5%
Into vacuum freeze drier drying 15 it is small when, obtain lyophilized Lumbrokinase.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
With within principle, any modifications, equivalent replacements and improvements are made should all be included in the protection scope of the present invention god.
Claims (7)
1. the extracting method of earthworm protein and Lumbrokinase in a kind of earthworm, which is characterized in that include the following steps：
(1) fresh full earthworm living is taken, washed several times with water as much as possible discharges the dirt in its internal organ, then pulls out with filter
Paper inhales its excessive moisture as far as possible；
(2) phosphate buffer for adding in the 0.1mol/LpH7.8 of 4 DEG C of precoolings is homogenized 5 minutes in homogenizer, in 4 DEG C of refrigerators
Stand 4 it is small when, rear high speed centrifugation discards precipitation, obtains supernatant；
(3) taking supernatant reinforcing body ammonium sulfate, 4000r/min centrifuges 10min, collects supernatant, add up to 35% saturation degree
Solid ammonium sulfate stands overnight, high speed centrifugation up to 70% saturation degree, collects precipitation, dissolved afterwards with 0.1mol/L phosphate buffers
This sediment filters to obtain crude protein solution；
(4) crude protein solution is added into deionized water desalination, is concentrated into concentrate and is in neutrality (pH7.0), when enrichment reaches 5% into
Enter vacuum freeze drier drying 5-20 it is small when to get earthworm protein freeze-dried powder；
(5) phosphate buffer that earthworm protein freeze-dried powder is added in 0.1mol/LpH7.8 dissolves, and 20000da- is crossed at both ends
40000da ultrafiltration membranes obtain trapped fluid A and filtered solution B；
(6) trapped fluid A is added into deionized water desalination, is concentrated into concentrate and is in neutrality (pH7.0), enrichment enters when reaching 5%
When vacuum freeze drier drying 5-20 is small, lyophilized Lumbrokinase is obtained；
(7) crude protein solution obtained by step (3) is added in into protease hydrolyzed, is digested using RF assistance, cross 6000da ultrafiltration membranes,
By filtered solution plus deionized water desalination, it is concentrated into concentrate and is in neutrality (pH7.0), enrichment enters vacuum refrigeration when reaching 5%
When drying machine drying 5-20 is small, 6500da ubiquitin-like earthworm polypeptides are obtained.
2. the extracting method of earthworm protein and Lumbrokinase in a kind of earthworm according to claim 1, which is characterized in that step
(2) phosphate buffer described in can also add in the sodium chloride of 0.1%-0.5% or sodium citrate or disodium hydrogen phosphate or carbonic acid
Hydrogen sodium or aqueous sodium carbonate.
3. the extracting method of earthworm protein and Lumbrokinase in a kind of earthworm according to claim 1, which is characterized in that step
(3) high speed centrifugation uses rotating speed 15000-25000r/min, horizontal spiral centrifuge.
4. the extracting method of earthworm protein and Lumbrokinase in a kind of earthworm according to claim 1, which is characterized in that step
(5) ultrafiltration membrane can also be bag filter, and dialyzate is 0.02mol/L phosphate buffers.
5. the extracting method of earthworm protein and Lumbrokinase in a kind of earthworm according to claim 1, which is characterized in that step
(5) filler of ultrafiltration membrane cartridge described in is one kind in glucan or agarose.
6. the extracting method of earthworm protein and Lumbrokinase in earthworm according to claim 1, which is characterized in that step (7)
Described in protease refer to neutral proteinase and trypsase.
7. the extracting method of earthworm protein and Lumbrokinase in a kind of earthworm according to claim 1, which is characterized in that described
The extracting method of earthworm protein and Lumbrokinase, subsidiary conditions are the radio frequency system of frequency 10MHz-200MHz.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110540550A (en) * | 2019-07-31 | 2019-12-06 | 倪成 | combined preparation process of lumbrokinase and crude earthworm lecithin |
| CN110592052A (en) * | 2019-09-22 | 2019-12-20 | 内蒙古新宏生物科技有限公司 | Production method and application of lumbruse polypeptide antibacterial nutrient solution |
| CN110606880A (en) * | 2019-07-31 | 2019-12-24 | 倪成 | Combined preparation process of earthworm elastin and lumbrokinase |
| CN110835625A (en) * | 2019-10-21 | 2020-02-25 | 倪成 | Production process of lumbrokinase concentrated slurry capable of being stored in normal temperature environment and transported for long distance |
| CN115772511A (en) * | 2022-12-22 | 2023-03-10 | 南开大学 | Method for efficiently extracting full resources from lumbrokinase |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110540550A (en) * | 2019-07-31 | 2019-12-06 | 倪成 | combined preparation process of lumbrokinase and crude earthworm lecithin |
| CN110606880A (en) * | 2019-07-31 | 2019-12-24 | 倪成 | Combined preparation process of earthworm elastin and lumbrokinase |
| CN110606880B (en) * | 2019-07-31 | 2021-11-09 | 倪成 | Combined preparation process of earthworm elastin and lumbrokinase |
| CN110592052A (en) * | 2019-09-22 | 2019-12-20 | 内蒙古新宏生物科技有限公司 | Production method and application of lumbruse polypeptide antibacterial nutrient solution |
| CN110592052B (en) * | 2019-09-22 | 2020-09-29 | 内蒙古新宏生物科技有限公司 | Production method and application of lumbruse polypeptide antibacterial nutrient solution |
| CN110835625A (en) * | 2019-10-21 | 2020-02-25 | 倪成 | Production process of lumbrokinase concentrated slurry capable of being stored in normal temperature environment and transported for long distance |
| CN115772511A (en) * | 2022-12-22 | 2023-03-10 | 南开大学 | Method for efficiently extracting full resources from lumbrokinase |
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Application publication date: 20180518 |