CN110408608B - Method for extracting lumbrokinase - Google Patents
Method for extracting lumbrokinase Download PDFInfo
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- CN110408608B CN110408608B CN201910775673.7A CN201910775673A CN110408608B CN 110408608 B CN110408608 B CN 110408608B CN 201910775673 A CN201910775673 A CN 201910775673A CN 110408608 B CN110408608 B CN 110408608B
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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- 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
- C12N9/6408—Serine endopeptidases (3.4.21)
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- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
Abstract
The invention relates to the field of lumbrokinase preparation, in particular to a lumbrokinase extraction method which mainly comprises the steps of water extraction, pre-separation, drying and crushing, and the steps are simple, a large amount of water can be extracted in a stirring tank, then the water is put into continuous separation equipment in batches, and the steps of filtering, concentrating and alcohol precipitating are carried out in the continuous separation equipment.
Description
Technical Field
The invention relates to the field of lumbrokinase preparation, in particular to a lumbrokinase extraction method.
Background
Lumbrukinase has proved to have better clinical application value, is a multi-molecular recombinant oral preparation, has special affinity with thrombus (fibrin), can track thrombolysis, effectively dissolve the thrombus, improve microcirculation, strengthen collateral circulation of heart and cerebral vessels, open repair of damaged endothelial cells of blood vessels, increase elasticity of blood vessels, improve oxygen supply function of blood vessels, reduce blood viscosity, reduce platelet aggregation rate and inhibit the reformation of thrombus. Repairing peripheral necrotic brain cells after thrombus occurs, and saving a half dark area. It is widely used in clinic and is increasingly used in the centers of prevention and treatment of diseases of heart, cerebral vessels, endocrine, respiratory system, etc.
The traditional decoction extraction method denatures most of the effective components and loses activity.
Because lumbrokinase has a plurality of important medicinal values and wide raw material sources, people continuously research the extraction method of lumbrokinase to finally obtain the extraction method of a plurality of lumbrokinase, which are listed as the following,
in order to improve the activity of lumbrokinase, the invention patent application with the application number of 201110103216.7 discloses a preparation method of high-activity lumbrokinase, which mainly comprises the steps of cleaning, precipitating, ultrafiltering, salting out, ion exchange and the like, and lumbrokinase with the enzyme activity of more than 40000U/mg is obtained;
in order to obtain lumbrokinase and earthworm protein, the invention patent application with the application number of 201711462748.3 discloses a method for extracting earthworm protein and lumbrokinase from earthworms, which mainly comprises the steps of water washing, homogenate, precipitation, salting out, concentration, ultrafiltration, concentration, enzymolysis, ultrafiltration and the like, so that lumbrokinase and 6500da earthworm polypeptide are obtained;
in order to obtain low-immunity lumbrokinase, the invention patent application with the application number of 201210123955.7 discloses a low-immunogenicity lumbrokinase and a preparation method and application thereof, firstly, a crude lumbrokinase is extracted from earthworms, then the crude lumbrokinase is eluted and purified by an ion exchange column, a component with fibrinolytic activity is collected, freeze-drying and concentration are carried out, a concentrated lumbrokinase solution is separated by a gel filtration chromatographic column, the fibrinolytic activity is detected by an agarose-fibrin plate method, a second elution peak with the fibrinolytic activity is collected, the molecular weight of the second elution peak is detected by SDS-PAGE electrophoresis, the second elution peak is 27500Da, the elution peak is subjected to dialysis, desalination, low-temperature freeze drying and molecular modification by adopting activated monomethoxypolyethylene glycol and dextran, and the lumbrokinase with the activity of 31400IU/mg can be obtained;
the three methods all obtain the lumbrukinase by respective technical schemes, can improve the quality of the lumbrukinase, but have complex preparation steps, high equipment cost used in the preparation process and low yield.
In order to reduce the production cost and ensure the yield of lumbrokinase, the invention patent application with the application number of 200910231486.9 discloses a preparation process method of lumbrokinase, which mainly comprises the steps of cleaning, grinding into slurry, removing slag, salting out, centrifuging, ultrafiltering, column chromatography separation, ultrafiltering, filtering, sterilizing, spraying and drying and the like.
The technical scheme provided by the invention with the application number of 200910231486.9 still has complex preparation steps and low production efficiency.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the lumbrokinase extraction method which is low in preparation cost, high in production efficiency and simple in preparation steps.
The invention is realized by the following technical scheme: a method for extracting lumbrokinase comprises the following steps,
step one, water extraction, namely putting earthworm powder into a stirring tank, adding drinking water with the amount 15 times that of the earthworm for the first time at normal temperature, starting stirring, stopping stirring after stirring for 15-20min, soaking for 3 h, adding drinking water with the amount 10 times that of the earthworm for the second time, stirring for 15-20min, soaking and standing for 8 h to obtain earthworm water extraction mixed solution;
step two, pre-separation, namely putting the earthworm water extraction mixed liquor obtained in the step one into continuous separation equipment for filtering, concentrating and alcohol precipitating to obtain pre-separation liquor;
step three, separation, namely putting the pre-separation liquid obtained in the step two into a centrifugal machine, and centrifuging to obtain lower-layer centrifugal paste;
drying, namely drying the lower-layer centrifugal paste obtained in the step three to obtain a lumbrokinase extract;
step five, crushing, namely crushing the lumbrukinase extract obtained in the step four to obtain finished lumbrukinase,
in the second step, pre-separation, the earthworm water extraction mixed liquor obtained in the first step is put into continuous separation equipment for filtration, concentration and alcohol precipitation to obtain pre-separated liquor, which comprises the following steps,
a, putting the earthworm water extraction mixture into a storage tank, draining the earthworm water extraction mixture in the storage tank through a pump through a filtering pipeline with a filter screen, and filtering to obtain clear solution containing lumbrukinase;
b, draining the clear solution containing the lumbrukinase to a transverse concentration tank by a pump, controlling the air pressure in the concentration tank to be-0.08-0.1 MPa by the pump, and introducing steam for concentration to obtain a concentrated solution with the density of 1.1-1.2 g/ml;
c, draining the concentrated solution into a transverse alcohol precipitation tank through a pump, adding ethanol, and allowing the concentrated solution to flow from an inlet end to an outlet end of the alcohol precipitation tank to obtain a pre-separation solution;
d, introducing the alcohol precipitation solution into a temporary storage tank through a pump for later use.
Further, in the first step, the temperature is controlled to be 23-30 ℃, and the stirring speed is 90-120 rpm.
Further, the centrifugal machine in the third step is a tubular centrifugal machine, and the rotating speed is 11000 and 13000 rpm.
Further, the temperature is controlled to be 10-15 ℃ in the fourth step.
Further, in the step a, the filter screen is a 100-mesh filter screen.
Further, the continuous separation equipment comprises a base, a storage tank is arranged on the base, a feeding port is arranged at the upper end of the storage tank, a gas port is further arranged at the upper end of the storage tank, a filter is arranged at the gas port, a liquid outlet pipe is arranged at the lower end of the storage tank, a filtering pipeline with a filter screen is communicated at the outlet of the liquid outlet pipe, the outlet end of the filtering pipeline is communicated with a transverse concentration tank through a first pipeline, a first check valve pointing to the concentration tank is arranged on the first pipeline, a steam generating device is connected to the upper side of the inlet end of the concentration tank, the outlet end of the concentration tank is communicated with the transverse alcohol precipitation tank through a pipeline, an ethanol tank is communicated with the inlet end of the alcohol precipitation tank, a circulating pump is connected between the inlet end and the outlet end of the alcohol precipitation tank, a circulating pipe is arranged at the outlet end of the circulating pipe, and the distance value, the utility model discloses a vacuum pump, including the alcohol precipitation jar, the exit end downside intercommunication of alcohol precipitation jar has the discharging pipe, be provided with the gear pump on the discharging pipe, the gear pump is to concentrated jar and alcohol precipitation jar negative pressure of taking out, the exit end of gear pump communicates to the temporary storage case through the pipeline, the installation height value of temporary storage case is higher than the gear pump, the bottom of temporary storage case is provided with the blowing pipe, temporary storage case lower extreme still is provided with the inspection pipe that has the valve, be provided with the sample frame on the base of inspection pipe downside.
Furthermore, a filtering device for filtering material slag is arranged on the filtering pipeline, the filtering device comprises a filtering box body, a filtering screen inserting opening is arranged on the filtering box body, a filtering screen frame is detachably inserted at the filtering screen inserting opening and comprises a bottom plate positioned outside the filtering box body, 2-3 filtering screen frames which are parallel to each other are arranged on the lower end face of the bottom plate, the filtering screen is arranged in the filtering screen frames, a blocking plate corresponding to the filtering screen frames is arranged at the filtering screen inserting opening, a sealing ring is arranged at the position, corresponding to the blocking plate, on the filtering screen frame, a hanging ring is arranged on the upper end face of the bottom plate, upward screw rods are arranged at the two sides of the filtering screen inserting opening at the upper end of the filtering box body, a cross rod for downwards pressing the bottom plate is arranged between the two corresponding screw rods, the two ends of the cross rod are penetrated on the corresponding screw rods, and, the bottom plate lower end face is provided with a sealing ring used for sealing with the filter screen box body, a downward slag discharge port is arranged at the inlet side of the lower end of the filter box body, which is located at the filter screen insertion port, a slag discharge pipe with a valve is connected to the lower side of the slag discharge port, the lower end of the slag discharge pipe is communicated with a slag collection tank, and the lower end of the slag collection tank is provided with a slag removal pipe with a valve.
Further, the steam generating device comprises a box body arranged on the base, a circulating fan is arranged in the box body, an air inlet of the circulating fan is communicated to the outlet end of the concentration tank, a steam generating box is further arranged in the box body, an electric heating tube is arranged in the steam generating box, an air outlet pipe of the steam generating box is communicated to the inlet end of the concentration tank, an air inlet pipe of the steam generating box is communicated with an air outlet of the circulating fan, a liquid discharging pipe with a valve is arranged at the lower end of the steam generating box, and a barometer is arranged on the concentration tank.
Further, the sample frame is including setting up the stand on the base, the stand upper end is provided with the swivel mount that is used for placing the sample test tube, the swivel mount includes the test tube backup pad that sets up with the stand coaxial line, it evenly is provided with a plurality of test tube jacks around the stand to fit in the test tube backup pad, be provided with the layer board that is used for lifting the test tube on the stand of test tube backup pad downside, layer board and test tube backup pad are the plectane, layer board and test tube backup pad all are through bearing coaxial line suit on the stand, be fixed with the spliced pole between layer board and the test tube backup pad, the layer board upper surface is provided with the cushion.
Further, the storage tank is a vertical storage tank, and a fixing frame for supporting the storage tank is arranged on the base.
The invention has the beneficial effects that: the method for extracting lumbrokinase mainly comprises the steps of water extraction, pre-separation, drying and crushing, and has simple steps, the method has the advantages that a large amount of water can be extracted in the stirring tank, then the water is put into the continuous separation equipment in batches, and the steps of filtering, concentrating and alcohol precipitating are carried out in the continuous separation equipment, only ethanol and steam are introduced in the whole extraction method, the cost is low, except the continuous separation equipment, only the stirring tank, the centrifugal machine, the drying equipment and the crushing equipment are needed, the operation is convenient, the intermediate links are few, the introduction of bacteria can be effectively avoided, the quality of lumbrokinase is ensured, in the continuous separation equipment, the filtering, concentrating and alcohol precipitating are integrated on a base, the adjustment and the connection are convenient, meanwhile, the control and maintenance are convenient, the water extract is put into the storage tank, and the pre-separation liquid subjected to alcohol precipitation is obtained from the water extract to the temporary storage tank, so that the transportation is not needed, the operation is convenient, and the production efficiency is improved.
Drawings
FIG. 1 is a schematic view showing the connection relationship of the respective parts of the continuous separation apparatus in example 1;
FIG. 2 is a schematic front sectional view of a filter device;
FIG. 3 is an enlarged view of a portion A of FIG. 2;
FIG. 4 is a schematic diagram of a screen port configuration;
FIG. 5 is a schematic diagram of a steam generator;
wherein: 1-base, 2-supporting legs, 3-fixing frame, 4-storage tank, 5-filter, 6-end cover, 7-valve, 8-filtering box, 9-slag collecting tank, 10-concentrating tank, 11-box, 12-barometer, 13-ethanol tank, 14-bracket, 15-circulation outlet pipe, 16-alcohol precipitation tank, 17-circulation pump, 18-one-way valve I, 19-gear pump, 20-temporary storage tank, 21-discharge pipe, 22-upright post, 23-inspection pipe, 24-test tube supporting plate, 25-supporting plate, 26-test tube, 27-connecting post, 28-discharge pipe, 29-slag removing pipe, 30-filter screen inserting port, 31-bottom plate, 32-sealing ring and 33-screw rod, 34-cross bar, 35-nut, 36-filter screen frame, 37-filter screen, 38-hoisting ring, 39-frame, 40-slot, 41-circulating fan, 42-steam generating box, 43-electric heating tube, 44-sealing ring.
Detailed Description
In the description of the present invention, it should also be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly and may, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the following embodiments, the continuous separation apparatus is shown in fig. 1-5, and comprises a base 1, the base 1 is constructed by welding h-shaped steel, the stability is high, the weight is light, the lower end of the base 1 is welded with supporting legs 2, the supporting legs are conveniently connected with a foundation, a storage tank 4 is installed on the base 1 through a fixing frame 3, the storage tank is a vertical storage tank, the capacity is 3 cubic meters, the storage tank is made of stainless steel material, a feeding port is processed at the upper end of the storage tank, an end cover 6 is arranged at the feeding port, an air port is also processed at the upper end of the storage tank, a filter 5 is installed at the air port, bacteria and dust can be filtered through the filter, so as to avoid polluting lumbrokinase, a liquid outlet pipe is installed at the lower end of the storage tank, a filter pipeline with a filter screen is communicated at the outlet of the liquid, the filter box body is provided with a filter screen inserting opening 30, a filter screen frame is detachably inserted at the filter screen inserting opening, specifically, the filter screen inserting opening comprises a rectangular frame 39, 3 mutually parallel inserting grooves 40 are processed on the frame, the filter screen frame comprises a bottom plate 31 positioned outside the filter box body, 3 mutually parallel filter screen frames 36 are fixed on the lower end surface of the bottom plate in a sealing way, a filter screen 37 is installed in each filter screen frame, each filter screen frame is made of stainless steel materials and has a thickness value of 3mm, inserting grooves for inserting filter screens are processed in each filter screen frame, each filter screen frame corresponds to 2 layers with a mesh number of 100, the overlapped rear edges of the two filter screen layers are bonded and then inserted in a groove, the filter screen frames are fixed through resin glue, volatile substances in the resin glue are released through aging treatment for 20-22h at 40-50 ℃, lumbrukinase pollution is avoided, a blocking plate corresponding to the filter screen frames is installed at the filter screen inserting opening, a slot for inserting a filter screen frame is formed between two adjacent barrier plates, a sealing ring 44 is adhered to the position, corresponding to the barrier plates, of the filter screen frame, the sealing ring is a natural rubber ring, a hanging ring 38 is installed on the upper end face of the bottom plate, a filter screen frame is convenient to move and is a vulnerable part, and the filter screen frame needs to be checked and replaced regularly, upward screw rods 33 are welded to two sides of an insertion opening of the filter screen at the upper end of the filter box body, four screw rods are distributed in a rectangular shape, cross rods 34 for pressing the bottom plate downwards are installed between the two corresponding screw rods, two ends of each cross rod are installed on the corresponding screw rods in a penetrating mode, nuts 35 are sleeved on the upper sides of the cross rods on the screw rods, the nuts are screwed, the cross rods push the bottom plate downwards, and further the bottom plate and the filter box body are pressed tightly, the disassembly, the replacement, the lower end of the filter box body is provided with a downward slag discharge port at the inlet side of the filter screen insertion port, the lower side of the slag discharge port is connected with a slag discharge pipe with a valve, the lower end of the slag discharge pipe is communicated with a slag collection tank 9, and the lower end of the slag collection tank is provided with a slag removal pipe 29 with a valve 7.
The outlet end of the filter pipeline is communicated to a transverse concentration tank 10 through a first pipeline, a first check valve 18 pointing to the concentration tank is installed on the first pipeline, a steam generating device is connected to the upper side of the inlet end of the concentration tank and comprises a box body 11 installed on a base 1, a circulating fan 41 is installed in the box body and is a Roots fan, the air inlet of the circulating fan is communicated to the outlet end of the concentration tank, a steam generating box 42 is further installed in the box body, an electric heating pipe 43 is installed in the steam generating box, the air outlet pipe of the steam generating box is communicated to the inlet end of the concentration tank, the air inlet pipe of the steam generating box is communicated with the air outlet of the circulating fan, a liquid discharging pipe 28 with a valve is installed at the lower end of the steam generating box, a barometer 12 is installed on the concentration tank, steam can be generated in the steam generating box through the, along with the continuation of concentrated step, the water in the steam generation incasement increases, regularly discharges through the casting pipe, can guarantee that concentrated jar normal work, owing to at the in-process of using, be the negative pressure in the concentrated jar, need use pump and casting pipe intercommunication in the use discharges water.
The outlet end of the concentration tank is communicated with a transverse alcohol precipitation tank 16 through a pipeline, the inlet end of the alcohol precipitation tank is communicated with an ethanol tank 13, a circulating pump is connected between the inlet end and the outlet end of the alcohol precipitation tank, a circulating outlet pipe is installed at the outlet end of the alcohol precipitation tank, the distance value between the inlet end of the circulating outlet pipe and the lower bottom surface of the alcohol precipitation tank is 1/3 of the total height value of the alcohol precipitation tank, 95% of ethanol can be supplemented into the alcohol precipitation tank through the ethanol tank, supernatant liquid at the outlet end of the alcohol precipitation tank can be pumped to the inlet end of the alcohol precipitation tank through the circulating pump, the using amount of the ethanol can be reduced, a discharging pipe is communicated with the lower side of the outlet end of the alcohol precipitation tank, a gear pump 19 is installed on the discharging pipe, the gear pump pumps negative pressure of the concentration tank and the alcohol precipitation tank, the outlet end of, the temporary storage box is arranged at a high position, the spraying can be avoided, the temporary storage box is convenient to take out, the discharging pipe 21 is arranged at the bottom of the temporary storage box, the pretreatment liquid in the temporary storage box can be taken out through the discharging pipe, in order to be convenient for extracting the pretreatment liquid for detection, the lower end of the temporary storage box is also provided with the inspection pipe 23 with a valve, the base 1 at the lower side of the inspection pipe is provided with the sampling frame, the sampling frame comprises a stand column 22 arranged on the base 1, the upper end of the stand column is provided with a rotating frame for placing a sampling test pipe 26, the rotating frame comprises a test pipe supporting plate 24 coaxially arranged with the stand column, a plurality of test pipe jacks are uniformly processed on the test pipe supporting plate around the stand column, the stand column at the lower side of the test pipe supporting plate is provided with a supporting plate 25 for supporting the test pipe, the supporting plate and the test, guarantee that layer board and test tube backup pad rotate in step before, surface mounting has the cushion on the layer board, and the cushion is the non-woven fabric pad, avoids test tube and layer board rigidity contact, can make the test tube just to the inspection tube sample through rotatory sample frame, and rotatory sample frame takes out the test tube after the sample.
The residue collection tank, the tank body, the circulating pump, the concentration tank, the alcohol precipitation tank, the ethanol tank, the temporary storage tank and the base 1 are all fixed through a support 14.
After the installation is finished, a one-way valve I pointing to the filter tank is installed between the concentration tank and the storage tank, in the using process, 75% of ethanol is firstly introduced, the continuous separation equipment is disinfected, then distilled water is introduced for cleaning, the valve is closed, earthworm water extraction mixed liquor after water extraction is put into the storage tank, 95% of ethanol is put into the ethanol tank, distilled water is injected into the steam generation box, distilled water is injected into the concentration tank and the alcohol precipitation tank, the injection amount is 1/2 of the volumes of the two, then the corresponding valve is opened, the alcohol precipitation tank and the concentration tank are vacuumized through the gear pump until the indication value of the air pressure is-0.1 MPa, in the process, due to the action of the pressure difference, the one-way valve I opens the corresponding valve, the circulating fan, the circulating pump and the electric heating pipe, the operation is started, the earthworm water extraction mixed liquor in the storage tank enters the concentration tank, and then, entering an alcohol precipitation tank for alcohol precipitation, finally entering a temporary storage box, emptying materials in the temporary storage box at intervals of 3min, sampling simultaneously, carrying out centrifugal precipitation after sampling, detecting precipitates, adjusting the power of an electric heating tube, adjusting the ethanol flow rate and adjusting the flow rate of the earthworm water extraction mixed solution by adjusting the rotating speed of a gear pump, adjusting the power of the electric heating tube, adjusting the ethanol flow rate and adjusting the flow rate of the earthworm water extraction mixed solution after the adjustment is finished every time, carrying out sampling detection, and recording the rotating speed of the gear pump, the power of the electric heating tube, the ethanol flow rate parameter and the flow rate of the earthworm water extraction mixed solution until the content of the precipitate lumbrokinase reaches a.
In the production process, the steam generating box needs to be drained periodically, the slag collecting tank needs to be discharged, and the filter screen needs to be replaced.
In the use process, because the two sides of the filter screen have pressure difference, the filter screen frame swings and is tightly pressed with the baffle plate through the sealing ring, thereby achieving better sealing effect.
Example 1
A method for extracting lumbrokinase comprises the following steps,
step one, water extraction, namely putting earthworm powder into a stirring tank, adding drinking water with the amount 15 times that of the earthworm for the first time at normal temperature, starting stirring, stopping stirring after stirring for 15min, soaking for 3 h, adding drinking water with the amount 10 times that of the earthworm for the second time, stirring for 18min, soaking and standing for 8 h to obtain earthworm water extraction mixed liquid, wherein the temperature is controlled at 24 ℃, the stirring speed is 90-120rpm, water extraction is carried out for 11 h, water-soluble lumbrokinase can be extracted into slurry, and purification is convenient;
step two, pre-separating, namely putting the earthworm water extraction mixed liquor obtained in the step one into continuous separation equipment for filtering, concentrating and alcohol precipitating to obtain a pre-separated liquor, wherein in the filtering process, the filter screen is a 100-mesh filter screen, dregs can be filtered, meanwhile, the slurry passes through the filter screen, lumbrukinase is extracted, in the concentrating process, the air pressure in a concentration tank is controlled to be 0.09MPa, the boiling point of the slurry under the air pressure is 25-35 ℃, evaporation can be accelerated on the premise of not influencing the activity of the lumbrukinase, the concentration efficiency is improved, the concentrated feed liquid density reaches 1.1-1.2g/ml, the concentration of the lumbrukinase is improved, then 95% ethanol is introduced, and the ethanol and the slurry are mixed to enable the concentration of the ethanol in the slurry to reach 69%, so that the pre-separated liquor is obtained;
step three, separation, namely putting the pre-separation liquid obtained in the step two into a centrifugal machine, and centrifuging to obtain a lower-layer centrifugal paste, wherein the centrifugal machine is a tubular centrifugal machine and the rotating speed is 11000 rpm;
drying, namely drying the lower-layer centrifugal paste obtained in the step three, wherein the temperature is controlled to be 12 ℃ during the drying process to obtain a lumbrokinase extract;
and step five, crushing, namely crushing the lumbrokinase extract obtained in the step four to obtain the finished lumbrokinase.
Lumbrokinase was prepared in this example, and the results are shown in attached table 1.
Example 2
A method for extracting lumbrokinase comprises the following steps,
step one, water extraction, namely putting earthworm powder into a stirring tank, adding drinking water with the amount 15 times that of the earthworm for the first time at normal temperature, starting stirring, stirring for 18min, then closing stirring, soaking for 3 h, adding drinking water with the amount 10 times that of the earthworm for the second time, stirring for 15min, soaking and standing for 8 h to obtain earthworm water extraction mixed liquid, wherein the temperature is controlled to be 25 ℃, the stirring speed is 100rpm, water extraction is carried out for 11 h, water-soluble lumbrokinase can be extracted into slurry, and purification is convenient;
step two, pre-separating, namely putting the earthworm water extraction mixed liquor obtained in the step one into continuous separation equipment for filtering, concentrating and alcohol precipitating to obtain a pre-separated liquor, wherein in the filtering process, the filter screen is a 100-mesh filter screen, dregs can be filtered, meanwhile, the serous fluid passes through the filter screen, and lumbrukinase is extracted, in the concentrating process, the air pressure in a concentrating tank is controlled to be 0.08-0.1MPa, the boiling point of the serous fluid under the air pressure is 25-35 ℃, evaporation can be accelerated on the premise of not influencing the activity of the lumbrukinase, the concentrating efficiency is improved, the density of the concentrated feed liquid reaches 1.1-1.2g/ml, the concentration of the lumbrukinase is improved, then 95% ethanol is introduced, and the ethanol and the serous fluid are mixed to enable the concentration of the ethanol in the serous fluid to reach 68-73%, so that the pre;
step three, separation, namely putting the pre-separation liquid obtained in the step two into a centrifugal machine, and centrifuging to obtain a lower-layer centrifugal paste, wherein the centrifugal machine is a tubular centrifugal machine and the rotating speed is 12000 rpm;
drying, namely drying the lower-layer centrifugal paste obtained in the step three, wherein the temperature is controlled to be 13 ℃ during the drying process to obtain a lumbrokinase extract;
and step five, crushing, namely crushing the lumbrokinase extract obtained in the step four to obtain the finished lumbrokinase.
Lumbrokinase was prepared in this example, and the results are shown in attached table 1.
Example 3
A method for extracting lumbrokinase comprises the following steps,
step one, water extraction, namely putting earthworm powder into a stirring tank, adding drinking water with the amount 15 times that of the earthworm for the first time at normal temperature, starting stirring, stopping stirring after stirring for 15min, soaking for 3 h, adding drinking water with the amount 10 times that of the earthworm for the second time, stirring for 20min, soaking and standing for 8 h to obtain earthworm water extraction mixed liquid, wherein the temperature is controlled at 30 ℃, the stirring speed is 120rpm, water extraction is carried out for 11 h, water-soluble lumbrokinase can be extracted into slurry, and purification is convenient;
step two, pre-separating, namely putting the earthworm water extraction mixed liquor obtained in the step one into continuous separation equipment for filtering, concentrating and alcohol precipitating to obtain a pre-separated liquor, wherein in the filtering process, the filter screen is a 100-mesh filter screen, dregs can be filtered, meanwhile, the serous fluid passes through the filter screen, and lumbrukinase is extracted, in the concentrating process, the air pressure in a concentrating tank is controlled to be 0.08-0.1MPa, the boiling point of the serous fluid under the air pressure is 25-35 ℃, evaporation can be accelerated on the premise of not influencing the activity of the lumbrukinase, the concentrating efficiency is improved, the density of the concentrated feed liquid reaches 1.1-1.2g/ml, the concentration of the lumbrukinase is improved, then 95% ethanol is introduced, and the ethanol and the serous fluid are mixed to enable the concentration of the ethanol in the serous fluid to reach 68-73%, so that the pre;
step three, separation, namely putting the pre-separation liquid obtained in the step two into a centrifugal machine, and centrifuging to obtain a lower-layer centrifugal paste, wherein the centrifugal machine is a tubular centrifugal machine and the rotating speed is 11000 rpm;
drying, namely drying the lower-layer centrifugal paste obtained in the step three, wherein the temperature is controlled to be 12 ℃ during the drying process to obtain a lumbrokinase extract;
and step five, crushing, namely crushing the lumbrokinase extract obtained in the step four to obtain the finished lumbrokinase.
Lumbrokinase was prepared in this example, and the results are shown in attached table 1.
Example 4
A method for extracting lumbrokinase comprises the following steps,
step one, water extraction, namely putting earthworm powder into a stirring tank, adding drinking water with the amount 15 times that of the earthworm for the first time at normal temperature, starting stirring, stopping stirring after stirring for 20min, soaking for 3 h, adding drinking water with the amount 10 times that of the earthworm for the second time, stirring for 18min, soaking and standing for 8 h to obtain earthworm water extraction mixed liquid, wherein the temperature is controlled at 27 ℃, the stirring speed is 110rpm, water extraction is carried out for 11 h, water-soluble lumbrokinase can be extracted into slurry, and purification is convenient;
step two, pre-separating, namely putting the earthworm water extraction mixed liquor obtained in the step one into continuous separation equipment for filtering, concentrating and alcohol precipitating to obtain a pre-separated liquor, wherein in the filtering process, the filter screen is a 100-mesh filter screen, dregs can be filtered, meanwhile, the serous fluid passes through the filter screen, and lumbrukinase is extracted, in the concentrating process, the air pressure in a concentrating tank is controlled to be 0.08-0.1MPa, the boiling point of the serous fluid under the air pressure is 25-35 ℃, evaporation can be accelerated on the premise of not influencing the activity of the lumbrukinase, the concentrating efficiency is improved, the density of the concentrated feed liquid reaches 1.1-1.2g/ml, the concentration of the lumbrukinase is improved, then 95% ethanol is introduced, and the ethanol and the serous fluid are mixed to enable the concentration of the ethanol in the serous fluid to reach 68-73%, so that the pre;
step three, separation, namely putting the pre-separation liquid obtained in the step two into a centrifugal machine, and centrifuging to obtain a lower-layer centrifugal paste, wherein the centrifugal machine is a tubular centrifugal machine and the rotating speed is 12000 rpm;
drying, namely drying the lower-layer centrifugal paste obtained in the step three, wherein the temperature is controlled to be 10-15 ℃ during the drying process, so as to obtain a lumbrokinase extract;
and step five, crushing, namely crushing the lumbrokinase extract obtained in the step four to obtain the finished lumbrokinase.
Lumbrokinase was prepared in this example, and the results are shown in attached table 1.
Example 5
A method for extracting lumbrokinase comprises the following steps,
step one, water extraction, namely putting earthworm powder into a stirring tank, adding drinking water with the amount 15 times that of the earthworm for the first time at normal temperature, starting stirring, stopping stirring after stirring for 15min, soaking for 3 h, adding drinking water with the amount 10 times that of the earthworm for the second time, stirring for 20min, soaking and standing for 8 h to obtain earthworm water extraction mixed liquid, wherein the temperature is controlled at 26 ℃, the stirring speed is 110rpm, water extraction is carried out for 11 h, water-soluble lumbrokinase can be extracted into slurry, and purification is convenient;
step two, pre-separating, namely putting the earthworm water extraction mixed liquor obtained in the step one into continuous separation equipment for filtering, concentrating and alcohol precipitating to obtain a pre-separated liquor, wherein in the filtering process, the filter screen is a 100-mesh filter screen, dregs can be filtered, meanwhile, the serous fluid passes through the filter screen, and lumbrukinase is extracted, in the concentrating process, the air pressure in a concentrating tank is controlled to be 0.08-0.1MPa, the boiling point of the serous fluid under the air pressure is 25-35 ℃, evaporation can be accelerated on the premise of not influencing the activity of the lumbrukinase, the concentrating efficiency is improved, the density of the concentrated feed liquid reaches 1.1-1.2g/ml, the concentration of the lumbrukinase is improved, then 95% ethanol is introduced, and the ethanol and the serous fluid are mixed to enable the concentration of the ethanol in the serous fluid to reach 68-73%, so that the pre;
step three, separation, namely putting the pre-separation liquid obtained in the step two into a centrifugal machine, and centrifuging to obtain a lower-layer centrifugal paste, wherein the centrifugal machine is a tubular centrifugal machine and the rotating speed is 11000 rpm;
drying, namely drying the lower-layer centrifugal paste obtained in the step three, wherein the temperature is controlled to be 15 ℃ during the drying process to obtain a lumbrokinase extract;
and step five, crushing, namely crushing the lumbrokinase extract obtained in the step four to obtain the finished lumbrokinase.
Lumbrokinase was prepared in this example, and the results are shown in attached table 1.
Attached table 1
The detection results of the technical scheme provided by the patent application with the application number of 201110103216.7 in the comparative example 1, the detection results of the technical scheme provided by the patent application with the application number of 201711462748.3 in the comparative example 2, and the detection results of the technical scheme provided by the patent application with the application number of 201210123955.7 in the comparative example 3 are shown in the attached table 1, and the lumbrokinase extraction method provided by the application can improve the yield of lumbrokinase to 12%.
The lumbrokinase prepared by the lumbrokinase extraction method provided by the application has high yield, the enzyme activity of the lumbrokinase can reach more than 10000U/mg, and the lumbrokinase can be widely applied to preparation of medicines and has wide market application prospect.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments or portions thereof without departing from the spirit and scope of the invention.
Claims (6)
1. A method for extracting lumbrokinase comprises the following steps,
step one, water extraction, namely putting earthworm powder into a stirring tank, adding drinking water with the amount 15 times that of the earthworm for the first time at normal temperature, starting stirring, stopping stirring after stirring for 15-20min, soaking for 3 h, adding drinking water with the amount 10 times that of the earthworm for the second time, stirring for 15-20min, soaking and standing for 8 h to obtain earthworm water extraction mixed solution;
step two, pre-separation, namely putting the earthworm water extraction mixed liquor obtained in the step one into continuous separation equipment for filtering, concentrating and alcohol precipitating to obtain pre-separation liquor;
step three, separation, namely putting the pre-separation liquid obtained in the step two into a centrifugal machine, and centrifuging to obtain lower-layer centrifugal paste;
drying, namely drying the lower-layer centrifugal paste obtained in the step three to obtain a lumbrokinase extract;
step five, crushing, namely crushing the lumbrukinase extract obtained in the step four to obtain finished lumbrukinase,
it is characterized in that in the second step, pre-separation is carried out, the earthworm water extraction mixed solution obtained in the first step is put into continuous separation equipment for filtration, concentration and alcohol precipitation, and the pre-separation solution is obtained by the following steps,
a, putting the earthworm water extraction mixture into a storage tank, draining the earthworm water extraction mixture in the storage tank through a pump through a filtering pipeline with a filter screen, and filtering to obtain clear solution containing lumbrukinase;
b, draining the clear solution containing the lumbrukinase to a transverse concentration tank by a pump, controlling the air pressure in the concentration tank to be-0.08-0.1 MPa by the pump, and introducing steam for concentration to obtain a concentrated solution with the density of 1.1-1.2 g/ml;
c, draining the concentrated solution into a transverse alcohol precipitation tank through a pump, adding ethanol, and allowing the concentrated solution to flow from an inlet end to an outlet end of the alcohol precipitation tank to obtain a pre-separation solution;
d, introducing the alcohol precipitation solution into a temporary storage tank through a pump for later use,
the continuous separation equipment comprises a base, a storage tank is arranged on the base, a feeding port is arranged at the upper end of the storage tank, a gas port is further arranged at the upper end of the storage tank, a filter is arranged at the gas port, a liquid outlet pipe is arranged at the lower end of the storage tank, a filtering pipeline with a filter screen is communicated at the outlet of the liquid outlet pipe, the outlet end of the filtering pipeline is communicated to a transverse concentration tank through a first pipeline, a first check valve pointing to the concentration tank is arranged on the first pipeline, a steam generating device is connected to the upper side of the inlet end of the concentration tank, the outlet end of the concentration tank is communicated to the transverse alcohol precipitation tank through a pipeline, an ethanol tank is communicated with the inlet end of the alcohol precipitation tank, a circulating pump is connected between the inlet end and the outlet end of the alcohol precipitation tank, a circulating pipe is arranged at the outlet end of the circulating pipe, and the, the lower side of the outlet end of the alcohol precipitation tank is communicated with a discharge pipe, a gear pump is arranged on the discharge pipe, the gear pump pumps negative pressure to the concentration tank and the alcohol precipitation tank, the outlet end of the gear pump is communicated to a temporary storage tank through a pipeline, the installation height of the temporary storage tank is higher than that of the gear pump, a discharge pipe is arranged at the bottom of the temporary storage tank, a test pipe with a valve is further arranged at the lower end of the temporary storage tank, and a sampling frame is arranged on a base at the lower side of the test pipe,
the filter device comprises a filter box body, a filter screen inserting opening is formed in the filter box body, a detachable filter screen frame is inserted in the filter screen inserting opening, the filter screen frame comprises a bottom plate located on the outer side of the filter box body, 2-3 filter screen frames which are parallel to each other are arranged on the lower end face of the bottom plate, the filter screen is arranged in the filter screen frames, a blocking plate corresponding to the filter screen frames is arranged at the filter screen inserting opening, a sealing ring is arranged on the filter screen frames at the position corresponding to the blocking plate, a hanging ring is arranged on the upper end face of the bottom plate, upward screw rods are arranged on the two sides of the filter screen inserting opening at the upper end of the filter box body, a cross rod used for pressing the bottom plate downwards is arranged between the two corresponding screw rods, two ends of the cross rod penetrate through the corresponding screw rods, and nuts are sleeved on the screw rods, the lower end face of the bottom plate is provided with a sealing ring for sealing with the filter screen box body, the lower end of the filter box body, which is positioned at the inlet side of the filter screen insertion opening, is provided with a downward slag discharge opening, the lower side of the slag discharge opening is connected with a slag discharge pipe with a valve, the lower end of the slag discharge pipe is communicated with a slag collection tank, the lower end of the slag collection tank is provided with a slag cleaning pipe with a valve,
the steam generating device comprises a box body arranged on a base, a circulating fan is arranged in the box body, an air inlet of the circulating fan is communicated with the outlet end of a concentration tank, a steam generating box is also arranged in the box body, an electric heating tube is arranged in the steam generating box, an air outlet pipe of the steam generating box is communicated with the inlet end of the concentration tank, an air inlet pipe of the steam generating box is communicated with an air outlet of the circulating fan, a liquid discharging pipe with a valve is arranged at the lower end of the steam generating box, a barometer is arranged on the concentration tank,
the sample frame is including setting up the stand on the base, the stand upper end is provided with the swivel mount that is used for placing the sample test tube, the swivel mount includes the test tube backup pad that sets up with the stand coaxial line, it evenly is provided with a plurality of test tube jacks around the stand to fit on the test tube backup pad, be provided with the layer board that is used for lifting the test tube on the stand of test tube backup pad downside, the layer board is the plectane with the test tube backup pad, the layer board all is through bearing coaxial line suit on the stand with the test tube backup pad, be fixed with the spliced pole between layer board and the test tube backup pad, the layer board upper.
2. The lumbrokinase extraction method of claim 1, wherein in the first step, the temperature is controlled at 23-30 ℃ and the stirring speed is 90-120 rpm.
3. The lumbrokinase extraction method of claim 1, wherein the centrifuge in step three is a tubular centrifuge, and the rotation speed is 11000 and 13000 rpm.
4. The lumbrokinase extraction method of claim 1, wherein the temperature is controlled at 10-15 ℃ in the fourth step.
5. The method for extracting lumbrokinase as claimed in claim 1, wherein in step a, the sieve is 100 mesh.
6. The lumbrokinase extraction method of claim 1, wherein the storage tank is a vertical storage tank, and the base is provided with a fixing frame for supporting the storage tank.
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Application publication date: 20191105 Assignee: Tianshui Jianyou Pharmaceutical Co.,Ltd. Assignor: SANMENXIA SINOWAY PHARMACEUTICAL Co.,Ltd. Contract record no.: X2023980037628 Denomination of invention: A method for extracting Lumbrokinase Granted publication date: 20210101 License type: Common License Record date: 20230706 |