CN110606880B - Combined preparation process of earthworm elastin and lumbrokinase - Google Patents
Combined preparation process of earthworm elastin and lumbrokinase Download PDFInfo
- Publication number
- CN110606880B CN110606880B CN201910702225.4A CN201910702225A CN110606880B CN 110606880 B CN110606880 B CN 110606880B CN 201910702225 A CN201910702225 A CN 201910702225A CN 110606880 B CN110606880 B CN 110606880B
- Authority
- CN
- China
- Prior art keywords
- earthworm
- elastic
- collagen
- lumbrokinase
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N9/14—Hydrolases (3)
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a combined preparation process of earthworm elastic (collagen) protein and lumbrokinase, which takes earthworms as raw materials, thoroughly separates a shell layer from an elastic protein layer by a freeze etching method, collects the obtained earthworm exudate as slurry and can be used for producing the lumbrokinase; purifying the rest part by countercurrent suspension method to obtain earthworm elastic collagen, and decocting, decolorizing, separating with membrane, concentrating, and freeze drying. The invention improves the utilization rate of earthworms, has no additive component in the process of producing the earthworm elastic collagen and the lumbrokinase, improves the quality of the earthworm elastic (collagen) protein and the lumbrokinase, reduces the production cost of the earthworm elastic (collagen) protein and the lumbrokinase, has simple preparation process, does not influence the activity of the lumbrokinase, and is suitable for industrial production.
Description
Technical Field
The invention relates to the field of earthworms, in particular to a combined preparation process of earthworm elastic (collagen) protein and lumbrokinase.
Background
The ancient medicine carried earthworm has cold and non-toxic properties, and enters liver, lung and kidney meridians. Has the effects of dispelling pathogenic wind, relieving fever, promoting urination, diminishing inflammation, relieving asthma and activating collaterals; earthworm is a Chinese medicinal prescription with adjuvant, which is handed down by official and civilian, hundreds of thousands of earthworms and is used along ancient and modern times. Today, there are a large number of organizations and organizations that aim to bring earthworm efficacy into social productivity; around the research and development hotspots of earthworm medical health care, earthworm functional food and earthworm protein feed, an earthworm fine chemical industry science and technology gate is silently formed. The method is characterized in that the method centers on innovative practice of earthworm scale breeding in the agriculture and animal husbandry, and large and small 'earthworm salon' and 'earthworm industry alliance' civil organizations and entity enterprises are searched for real names in a network, and the method is based on top-down.
The method is a large industry and is self-scenery in the European and American earthworm industry in the aspects of expanding the agricultural and environment-friendly industrial markets. In China, the industrialization of the anti-thrombus drug lumbrokinase greatly promotes the development of earthworms in China, but the bottleneck limitations that the scale is small and the climate technology is lagged exist at the same time. From the national situation perspective, the technology of the earthworm industry is characterized in that: small earthworms, high technology, industrialization, large network industry, root-planting earthworm agriculture and earthworm environment protection. Wherein, the support of the high technology of earthworm fine chemical engineering is indispensable. However, the traditional preparation process can only extract a single product from the earthworms, which inevitably causes certain waste and causes low utilization rate of the earthworms.
The earthworm body contains a great amount of elastin and collagen, so that the body surface of the live earthworm can be kept wet forever, and the body length can be freely stretched and changed within the range of 4-5 times, so that the earthworm body is made to exist in the elastin and the collagen.
In the medical field, collagen determines the softness, moistness and fullness of skin, and elastin determines the tightness and elasticity of skin. The experimental evidence of the freeze-etching technology shows that elastin concentrates on the body wall of the earthworm, and in view of natural symbiosis of elastin and collagen and the fact that the elastin is not further separated intentionally in the process of extracting elastin, elastin and collagen in the patent are called as "elasticity (collagen) protein" or simplified as "elasticity collagen".
Disclosure of Invention
In view of the above, the present invention aims to provide a combined preparation process of earthworm elastic (collagen) protein and lumbrokinase, which can simultaneously extract lumbrokinase and improve the availability of earthworms.
In order to achieve the purpose, the invention provides the following technical scheme:
a process for preparing earthworm elastic (collagen) protein and lumbrokinase in combination includes:
a) frozen etching
1) Quick freezing at low temperature: after the abdominal cavity impurities of the earthworms are emptied, the earthworms are cleaned and then placed at the temperature of minus 45 ℃ for 4 to 8 hours to obtain frozen earthworms;
2) and (3) thawing at constant temperature: transferring the frozen earthworms obtained in the step 1) to 70-80 ℃ for unfreezing, and collecting earthworm exudates;
b) separation of elastin by counter current suspension method
Carrying out countercurrent suspension separation on the substances left after the earthworm exudates are collected in the step a), and removing impurities to obtain earthworm elastin;
c) extracting earthworm elastic (collagen) protein
Adding deionized water into the earthworm elastin obtained in the step b), keeping the temperature in a water bath kettle at 80-90 ℃ for 2h, performing vacuum negative pressure suction filtration to obtain filtrate, and decoloring the filtrate to obtain earthworm elastin (collagen) liquid;
d) extracting and concentrating earthworm elastic (collagen) protein
Centrifuging and clarifying the earthworm elastic (collagen) protein liquid obtained in the step c) at 10000r/min, and grading and concentrating the clarified liquid through a hollow fiber column with the molecular weight cutoff of 6000-100000D to obtain an elastic (collagen) protein concentrated liquid;
e) freezing and drying the elastic (collagen) protein concentrate obtained in the step d) at the temperature of-75 ℃ and under the pressure of 10-15Pa to obtain earthworm elastic (collagen) protein;
f) extraction of lumbrukinase
Adjusting the pH value of the earthworm exudate collected in the step a) to 7.8 by using 0.005mol/L PBS buffer solution, concentrating by 5 times, performing separation and purification chromatography on the concentrated solution through anion exchange cellulose resin ion exchange chromatography, performing stage elution on a product after chromatography by using 0.5-2mol/L sodium chloride to obtain lumbrokinase lower column solution, and desalting, concentrating, and freeze-drying to obtain lumbrokinase.
Preferably, the step a) and the step 2) are repeated for 7-10 times, and earthworm exudates are collected and combined.
Preferably, in the step b), the temperature of the deionized water in the countercurrent suspension is 4-8 ℃.
Preferably, in the step c), the mass ratio of the earthworm elastin to the deionized water is 1: 10.
preferably, in step c), the filtrate is decolorized using activated carbon and/or anion exchange cellulose resin.
The invention discloses a combined preparation process of earthworm elastic (collagen) protein and lumbrokinase, which takes earthworms as raw materials, thoroughly separates a shell layer from an elastic protein layer by a freeze etching method, collects the obtained earthworm exudate as slurry and can be used for producing the lumbrokinase; purifying the rest part by countercurrent suspension method to obtain elastin, decocting, decolorizing, membrane separating, concentrating, and freeze drying to obtain Lumbricus elastic (collagen) protein. The invention improves the utilization rate of earthworms, has no additive component in the process of producing the earthworm elastic (collagen) protein and the lumbrokinase, improves the quality of the earthworm elastic (collagen) protein and the lumbrokinase, reduces the production cost of the earthworm elastic (collagen) protein and the lumbrokinase, has simple preparation process, does not influence the activity of the lumbrokinase, and is suitable for industrial production.
Drawings
FIG. 1 is a water retention test picture;
FIG. 2 is a photograph of the earthworm elastic force (collagen) protein prepared in example 3;
FIG. 3 is a diagram showing the effect of the earthworm elastic (collagen) protein decolouring;
FIG. 4 is a diagram of lumbrokinase potency assay.
Detailed Description
For a further understanding of the invention, preferred embodiments of the invention are described below in conjunction with the examples, but it should be understood that these descriptions are included merely to further illustrate the features and advantages of the invention and are not intended to limit the invention to the claims.
The invention provides a combined preparation process of earthworm elastic (collagen) protein and lumbrokinase, which comprises the following steps:
a) frozen etching
1) Quick freezing at low temperature: after the abdominal cavity impurities of the earthworms are emptied, the earthworms are cleaned and then placed at the temperature of minus 45 ℃ for 4 to 8 hours to obtain frozen earthworms;
2) and (3) thawing at constant temperature: transferring the frozen earthworms obtained in the step 1) to 70-80 ℃ for unfreezing, and collecting earthworm exudates;
b) separation of elastic (collagen) protein by counter-current suspension method
And c) carrying out countercurrent suspension separation on the substances remained after the earthworm exudates are collected in the step a), and removing impurities to obtain the earthworm elastic (collagen) protein.
c) Extracting earthworm elastic (collagen) protein
Adding deionized water into the earthworm elastin obtained in the step b), keeping the temperature in a water bath kettle at 80-90 ℃ for 2h, performing vacuum negative pressure suction filtration to obtain filtrate, and decoloring the filtrate to obtain earthworm elastin (collagen) liquid;
d) extracting and concentrating earthworm elastic (collagen) protein
Centrifuging and clarifying the earthworm elastic (collagen) protein liquid obtained in the step c) at 10000r/min, and grading and concentrating the clarified liquid through a hollow fiber column with the molecular weight cutoff of 6000-100000D to obtain an elastic (collagen) protein concentrated liquid;
e) freezing and drying the elastic (collagen) protein concentrate obtained in the step d) at the temperature of-75 ℃ and under the pressure of 10-15Pa to obtain earthworm elastic (collagen) protein;
f) extraction of lumbrukinase
Adjusting the pH value of the earthworm exudate collected in the step a) to 7.8 by using 0.005mol/L PBS buffer solution, concentrating by 5 times, performing separation and purification chromatography on the concentrated solution through anion exchange cellulose resin ion exchange chromatography, performing stage elution on a product after chromatography by using 0.5-2mol/L sodium chloride to obtain lumbrokinase lower column solution, and desalting, concentrating and freeze-drying to obtain lumbrokinase.
According to the technical scheme, earthworms are used as raw materials, a shell layer and an elastin layer are thoroughly separated by a freeze etching method, and the collected earthworm exudate is used as slurry and can be used for producing lumbrokinase; purifying the rest part by countercurrent suspension method to obtain elastin, decocting, decolorizing, membrane separating, concentrating, and freeze drying to obtain Lumbricus elastic (collagen) protein. The invention improves the utilization rate of earthworms, has no additive component in the process of producing the earthworm elastic (collagen) protein and the lumbrokinase, improves the quality of the earthworm elastic (collagen) protein and the lumbrokinase, reduces the production cost of the earthworm elastic (collagen) protein and the lumbrokinase, has simple preparation process, does not influence the activity of the lumbrokinase, and is suitable for industrial production.
In the invention, after the abdominal cavity sundries of the earthworms are emptied, the earthworms are cleaned and then placed at the temperature of minus 45 ℃ for 4 to 8 hours to obtain frozen earthworms; in the low-temperature quick-freezing step, the earthworm tissue cell serous fluid is quickly frozen, so that the cell wall is broken, and the lumbrokinase is released and the chitin is peeled.
Transferring the obtained frozen earthworms to 70-80 ℃ for unfreezing, and collecting earthworm exudates; rapidly thawing the frozen earthworms at the constant temperature of 70-80 ℃, seeping out liquid in the thawing process, and collecting the exudate; it should be noted that, in order to ensure the activity of lumbrokinase, the temperature of the earthworms in the whole thawing process must not exceed 40-45 ℃.
In the embodiment of the invention, low-temperature quick freezing and constant-temperature thawing are repeated for 7-10 times, and earthworm exudates are collected and combined; and (3) repeating the quick freezing and the unfreezing for 7-10 times, so that the earthworm elastin cortex is completely exposed, the peeled earthworm shell is used as a chitin raw material, and the exudate collected in the unfreezing process is used as a raw material of lumbrokinase.
The earthworm shells and the elastin cortex can be effectively stripped in the freezing and etching process, and can be thoroughly separated after 7-10 times of circulation, so that subsequent elastin can be conveniently collected.
Carrying out countercurrent suspension separation on the substances left after the earthworm exudates are collected, and removing impurities to obtain earthworm elastin; the step of separating the elastin by the countercurrent suspension method can strip the elastin (collagen) from the earthworm shell. It should be noted that the apparatus used in the countercurrent suspension method is high: diameter 1: 20-25 of cylinder, reversely introducing deionized water of 4-8 deg.C (i.e. introducing deionized water of 4-8 deg.C into the inlet at the lower end), controlling the flow rate to make elastin float and dregs not float, and collecting pure earthworm elastic (collagen) protein at the outlet at the upper end.
Adding deionized water into the earthworm elastic (collagen) protein, keeping the temperature in a water bath kettle at 80-90 ℃ for 2h, carrying out vacuum negative pressure suction filtration to obtain a filtrate, and decoloring the filtrate to obtain the earthworm elastic (collagen) protein liquid; in the step of extracting the earthworm elastic (collagen) protein, the earthworm elastic protein is kept warm in a water bath kettle for 2 hours, and the elastic (collagen) protein can be dissolved out; negative pressure suction filtration is used for removing deformed nucleic acid and foreign protein. In the embodiment of the invention, the mass ratio of the earthworm elastin to the deionized water is 1: 10; the filtrate is decolorized using activated carbon and/or anion exchange cellulose resin.
And (3) centrifuging and clarifying the earthworm elastic (collagen) protein liquid at 10000r/min, and classifying and concentrating the clarified liquid through a hollow fiber column with the cut-off molecular weight of 6000-100000D to obtain the elastic (collagen) protein concentrated liquid.
Freeze drying the elastic (collagen) protein concentrate at-75 deg.C under 10-15Pa to obtain Lumbricus elastic (collagen) protein. The earthworm elastic (collagen) protein is obtained by purifying the elastin by a countercurrent suspension method, boiling the glue, decoloring, separating a membrane, concentrating and freeze-drying, and has high water locking capacity and elastic modulus.
Adjusting the pH value of the collected earthworm exudate to 7.8 by using 0.005mol/L PBS buffer solution, concentrating by 5 times, performing separation and purification chromatography on the concentrated solution through anion exchange cellulose resin ion exchange chromatography, and performing stage elution on the chromatographed product by using 3mol/L sodium chloride to obtain lumbrokinase; the activity peak value of lumbrukinase extracted in the step reaches 106U/Ml。
In order to further illustrate the present invention, the combined preparation process of earthworm elastic (collagen) protein and lumbrokinase provided by the present invention is described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
a) Frozen etching
1) Quick freezing at low temperature: after the abdominal cavity impurities of the earthworms are emptied, the earthworms are cleaned and then placed at the temperature of minus 45 ℃ for 4 hours to obtain frozen earthworms;
2) and (3) thawing at constant temperature: transferring the frozen earthworms obtained in the step 1) to 70 ℃ for unfreezing, and collecting earthworm exudates;
3) repeating the steps 1) and 2) for 7 times, collecting and combining earthworm exudates, and collecting the rest parts of the earthworms;
b) separation of elastin by counter current suspension method
Carrying out countercurrent suspension separation on the substances left after the earthworm exudates are collected in the step a), wherein the temperature of deionized water is 4 ℃, and removing impurities to obtain earthworm elastin;
c) extracting earthworm elastic (collagen) protein
Adding deionized water into the earthworm elastin obtained in the step b), wherein the mass ratio of the earthworm elastin to the deionized water is 1: 10, keeping the temperature in a water bath kettle at 80 ℃ for 2 hours, performing vacuum negative pressure suction filtration to obtain filtrate, and decoloring the filtrate to obtain earthworm elastic (collagen) protein liquid;
d) extracting and concentrating earthworm elastic (collagen) protein
Centrifuging and clarifying the earthworm elastic (collagen) protein liquid obtained in the step c) at 10000r/min, and grading and concentrating the clarified liquid through a hollow fiber column with the molecular weight cutoff of 6000D to obtain an elastic (collagen) protein concentrated liquid;
e) freezing and drying the elastic (collagen) protein concentrate obtained in the step d) at the temperature of-75 ℃ and under the pressure of 10Pa to obtain earthworm elastic (collagen) protein;
f) extraction of lumbrukinase
Adjusting the pH value of the earthworm exudate collected in the step a) to 7.8 by using 0.005mol/L PBS buffer solution, concentrating by 5 times, performing separation and purification chromatography on the concentrated solution through anion exchange cellulose resin ion exchange chromatography, performing stage elution on a product after chromatography by using 2mol/L sodium chloride to obtain lumbrokinase lower column solution, and desalting, concentrating, and freeze-drying to obtain lumbrokinase.
Example 2
a) Frozen etching
1) Quick freezing at low temperature: after the abdominal cavity impurities of the earthworms are emptied, the earthworms are cleaned and then placed at the temperature of minus 45 ℃ for 8 hours to obtain frozen earthworms;
2) and (3) thawing at constant temperature: transferring the frozen earthworms obtained in the step 1) to 90 ℃ for unfreezing, and collecting earthworm exudates;
3) repeating the step 1) and the step 2) for 10 times, collecting and combining earthworm exudates, and collecting the rest parts of the earthworms;
b) separation of elastin by counter current suspension method
Carrying out countercurrent suspension separation on the substances left after the earthworm exudates are collected in the step a), and removing impurities to obtain earthworm elastin;
c) extracting earthworm elastic (collagen) protein
Adding deionized water into the earthworm elastic protein obtained in the step b), keeping the temperature in a water bath kettle at 90 ℃ for 2 hours, carrying out vacuum negative pressure suction filtration to obtain filtrate, and decoloring the filtrate to obtain earthworm elastic (collagen) protein liquid;
d) extracting and concentrating earthworm elastic (collagen) protein
Centrifuging and clarifying the earthworm elastic (collagen) protein liquid obtained in the step c) at 10000r/min, and grading and concentrating the clarified liquid through a hollow fiber column with the molecular weight cutoff of 100000D to obtain an elastic (collagen) protein concentrated liquid;
e) freezing and drying the elastic (collagen) protein concentrate obtained in the step d) at the temperature of-75 ℃ and under the pressure of 15Pa to obtain earthworm elastic (collagen) protein;
f) extraction of lumbrukinase
Adjusting the pH value of the earthworm exudate collected in the step a) to 7.8 by using 0.005mol/L PBS buffer solution, concentrating by 5 times, performing separation and purification chromatography on the concentrated solution through anion exchange cellulose resin ion exchange chromatography, performing stage elution on a product after chromatography by using 0.5mol/L sodium chloride to obtain lumbrokinase lower column solution, and desalting, concentrating, and freeze-drying to obtain lumbrokinase.
Example 3
a) Frozen etching
1) Quick freezing at low temperature: after the abdominal cavity impurities of the earthworms are emptied, the earthworms are cleaned and then placed at the temperature of minus 45 ℃ for 6 hours to obtain frozen earthworms;
2) and (3) thawing at constant temperature: transferring the frozen earthworms obtained in the step 1) to 75 ℃ for unfreezing, and collecting earthworm exudates;
3) repeating the step 1) and the step 2) for 8 times, collecting and combining earthworm exudates, and collecting the rest parts of the earthworms;
b) separation of elastin by counter current suspension method
Carrying out countercurrent suspension separation on the substances left after the earthworm exudates are collected in the step a), and removing impurities to obtain earthworm elastin;
c) extracting earthworm elastic (collagen) protein
Adding deionized water into the earthworm elastic protein obtained in the step b), keeping the temperature in a water bath kettle at 85 ℃ for 2 hours, carrying out vacuum negative pressure suction filtration to obtain filtrate, and decoloring the filtrate to obtain earthworm elastic (collagen) protein liquid;
d) extracting and concentrating earthworm elastic (collagen) protein
Centrifuging and clarifying the earthworm elastic (collagen) protein liquid obtained in the step c) at 10000r/min, and grading and concentrating the clarified liquid through a hollow fiber column with the cut-off molecular weight of 50000D to obtain an elastic (collagen) protein concentrated liquid;
e) the elasticity (collagen) obtained in the step d)
Freeze drying the protein concentrate at-75 deg.C under 15Pa to obtain Lumbricus elastic (collagen) protein;
f) extraction of lumbrukinase
Adjusting the pH value of the earthworm exudate collected in the step a) to 7.8 by using 0.005mol/L PBS buffer solution, concentrating by 5 times, performing separation and purification chromatography on the concentrated solution through anion exchange cellulose resin ion exchange chromatography, performing stage elution on a product after chromatography by using 1mol/L sodium chloride to obtain lumbrokinase lower column solution, and desalting, concentrating, and freeze-drying to obtain lumbrokinase.
The elastin obtained in example 1-3 was tested for elastic modulus and water retention.
Method for testing elastic modulus: two sections of earthworm elastin of equal length were taken from examples 1-3, one end was immobilized and the other end was forced, and the average elastic modulus values are shown in table 1.
TABLE 1 results of the experiment
| Mean modulus of elasticity | |
| Example 1 | 4.2 |
| Example 2 | 4.4 |
| Example 3 | 4.3 |
Testing of water retention: the earthworm elastin in examples 1-3 was taken out and dried, and it was found that the earthworm elastin in examples 1-3 all formed transparent films; and adding water into the dried earthworm elastin, so that the transparent diaphragm can be found to be rapidly swelled, and the original water locking capacity is recovered. Indicating that the physicochemical properties of the dried earthworm elastin remained intact. Specifically, as shown in fig. 1, the left image is a transparent film formed by dehydrating and drying the elastin, and the right image is a picture of the transparent film after the transparent film is rapidly swelled with water.
The extraction rates of earthworm elastic (collagen) protein and lumbrokinase obtained in examples 1 to 3 were measured, and the results are shown in Table 2.
TABLE 2 results of the experiment
The earthworm elastic (collagen) protein prepared in example 3 is shown in figure 2; to test the bleaching effect, the results are shown in FIG. 3. As can be seen from FIG. 3, the earthworm elastic (collagen) protein extracted by the invention has good decolorization effect. Wherein the left image is a photograph before decoloring and the right image is a photograph after decoloring.
Meanwhile, the lumbrokinase prepared in examples 1-3 is detected, the method is shown in figure 4, and the result is shown in table 3.
TABLE 3 results of the experiment
The present invention provides a method for preparing earthworm elastic (collagen) and lumbrokinase in combination, which is described in detail above, and the principle and embodiments of the present invention are illustrated herein by using specific examples, and the description of the above examples is only for assisting understanding of the method of the present invention and the core concept thereof, it should be noted that, for those skilled in the art, the present invention may be modified and modified without departing from the principle of the present invention, and the modified and modified aspects also fall within the protection scope of the claims of the present invention.
Claims (5)
1. A combined preparation process of earthworm elastic collagen and lumbrokinase is characterized by comprising the following steps:
a) frozen etching
1) Quick freezing at low temperature: after the abdominal cavity impurities of the earthworms are emptied, the earthworms are cleaned and then placed at the temperature of minus 45 ℃ for 4 to 8 hours to obtain frozen earthworms;
2) and (3) thawing at constant temperature: thawing the frozen earthworms obtained in the step 1) at 70-80 ℃, and collecting earthworm exudate;
b) separation of elastin by counter current suspension method
Carrying out countercurrent suspension separation on the substances left after the earthworm exudates are collected in the step a), and removing impurities to obtain earthworm elastin; the devices adopted by the countercurrent suspension method are high: diameter 1: 20-25 of cylinder;
c) extracting earthworm elastic collagen
Adding deionized water into the earthworm elastin obtained in the step b), keeping the temperature in a water bath kettle at 80-90 ℃ for 2h, performing vacuum negative pressure suction filtration to obtain filtrate, and decoloring the filtrate to obtain earthworm elastic collagen liquid;
d) extracting and concentrating earthworm elastic collagen
Centrifuging and clarifying the earthworm elastic collagen liquid obtained in the step c) at 10000r/min, and grading and concentrating the clarified liquid through a hollow fiber column with the molecular weight cutoff of 6000-100000D to obtain an elastic collagen concentrated liquid;
e) freezing and drying the elastic collagen concentrated solution obtained in the step d) at the temperature of-75 ℃ and under the pressure of 10-15Pa to obtain earthworm elastic collagen;
f) extraction of lumbrukinase
Adjusting the pH value of the earthworm exudate collected in the step a) to 7.8 by using 0.005mol/L PBS buffer solution, concentrating by 5 times, performing separation and purification chromatography on the concentrated solution through anion exchange cellulose resin ion exchange chromatography, performing stage elution on a product after chromatography by using 0.5-2mol/L sodium chloride, desalting, concentrating, and freeze-drying to obtain lumbrokinase.
2. The combined preparation process of claim 1, wherein in step a), the steps 1) and 2) are repeated for 7-10 times, and earthworm exudates are collected and combined.
3. The integrated preparation process according to claim 1, wherein in step b) the temperature of the deionized water in the countercurrent suspension is 4 to 8 ℃.
4. The integrated preparation process of claim 1, wherein in step c), the mass ratio of the earthworm elastin to the deionized water is 1: 10.
5. the integrated process of claim 1, wherein in step c) the filtrate is decolorized using activated carbon and/or anion exchange cellulose resin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910702225.4A CN110606880B (en) | 2019-07-31 | 2019-07-31 | Combined preparation process of earthworm elastin and lumbrokinase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910702225.4A CN110606880B (en) | 2019-07-31 | 2019-07-31 | Combined preparation process of earthworm elastin and lumbrokinase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110606880A CN110606880A (en) | 2019-12-24 |
| CN110606880B true CN110606880B (en) | 2021-11-09 |
Family
ID=68891030
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910702225.4A Active CN110606880B (en) | 2019-07-31 | 2019-07-31 | Combined preparation process of earthworm elastin and lumbrokinase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110606880B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111393522B (en) * | 2020-03-27 | 2021-04-23 | 华南师范大学 | Collagen and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1657098A (en) * | 2004-02-20 | 2005-08-24 | 北京百奥药业有限责任公司 | Preparation method of earthworm kinase dry powder |
| CN101717761A (en) * | 2009-11-24 | 2010-06-02 | 北京百奥药业有限责任公司 | Application of lumbrukinase in treating carotid atherosclerosis |
| CN102876652A (en) * | 2012-09-28 | 2013-01-16 | 北京百奥未来生物科技有限公司 | Dried lumbrukinase powder preparation method |
| CN108048434A (en) * | 2017-12-28 | 2018-05-18 | 天津百利食品有限公司 | The extracting method of earthworm protein and Lumbrokinase in a kind of earthworm |
-
2019
- 2019-07-31 CN CN201910702225.4A patent/CN110606880B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1657098A (en) * | 2004-02-20 | 2005-08-24 | 北京百奥药业有限责任公司 | Preparation method of earthworm kinase dry powder |
| CN101717761A (en) * | 2009-11-24 | 2010-06-02 | 北京百奥药业有限责任公司 | Application of lumbrukinase in treating carotid atherosclerosis |
| CN102876652A (en) * | 2012-09-28 | 2013-01-16 | 北京百奥未来生物科技有限公司 | Dried lumbrukinase powder preparation method |
| CN108048434A (en) * | 2017-12-28 | 2018-05-18 | 天津百利食品有限公司 | The extracting method of earthworm protein and Lumbrokinase in a kind of earthworm |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110606880A (en) | 2019-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102746412B (en) | Method for extracting momordica charantiap polysaccharide | |
| CN109527270A (en) | Leech specific nutrient induces the extracting method of liquid and natural hirudin | |
| CN107163162A (en) | A kind of refined method of LBP-X | |
| CN101120776A (en) | Method for extracting beta-glucan from cereal bran using membrane separation technology | |
| CN103961797A (en) | Method for promoting secretion of snail mucus and extraction method thereof | |
| CN111557893A (en) | Preparation method of mask containing stem cell exosome components | |
| CN110606880B (en) | Combined preparation process of earthworm elastin and lumbrokinase | |
| CN105039480A (en) | Method for purifying collagen derived from pelodiscus sinensis | |
| CN112980765A (en) | Method for extracting high-purity centella asiatica exosome | |
| CN104611316A (en) | Method for extracting bromelin | |
| CN105687036B (en) | Bletilla skin-protection product and preparation method thereof | |
| CN106478831A (en) | A kind of Paeonia suffruticosa polysaccharide and its preparation method and application | |
| CN110283257A (en) | A kind of extracting method of Radix Angelicae Sinensis polysaccharide | |
| CN104140403A (en) | Andrographolide separation and purification process | |
| CN102784193A (en) | Method for preparing hedysarum polybotrys extract by adopting coupling technology | |
| CN113061199A (en) | Process for concentrating and extracting crude heparin sodium by using nanofiltration membrane | |
| CN115975065B (en) | High-purity low-molecular-weight polygonatum polysaccharide PSP-1-1, polygonatum oligosaccharide PSO, and method and application thereof | |
| CN103724456B (en) | The Technology for normal-temperature salt-free extraction of heparin sodium | |
| CN102964407B (en) | Method for extracting astilbin from rhizoma smilacis glabrae and astilbin prepared thereby | |
| CN106928347A (en) | A kind of separation method of hirudin | |
| CN102552369A (en) | Lon icera japonica Thunb leaf extractive serving as anti-porcine reproductive and respiratory syndrome viral agent | |
| CN115521387A (en) | Acidic polysaccharide and method for separating acidic polysaccharide from basil seed gum | |
| CN113980155A (en) | Method for extracting heparin in animal lungs | |
| CN106389507A (en) | Bitter gourd membrane extraction method | |
| CN108191924A (en) | A kind of method from prawn shell extraction chondroitin sulfate |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |