Aqueous two-phase extraction device and method for simultaneously extracting ethanol dehydrogenase and heparin from pig liver

Abstract

This invention provides a two-phase aqueous extraction apparatus and a method for simultaneously extracting alcohol dehydrogenase and heparin from pig liver, belonging to the field of chemical manufacturing technology. The two-phase aqueous extraction apparatus includes two storage cylinders; each storage cylinder has an independently mounted rotating shaft; a driving wheel is fixedly mounted on each rotating shaft; a first driven wheel is mounted on the left side of the driving wheel; a first horizontal shaft is fixedly mounted at the center of the first driven wheel; the first horizontal shaft is rotatably mounted on the inner wall of the storage cylinder; a first stirring rod is evenly arranged on the outer wall of the first horizontal shaft; a second driven wheel is engaged on the right side of the driving wheel; a second horizontal shaft is fixedly mounted at the center of the second driven wheel. Using this apparatus to extract from pig liver is not only efficient, simple, and environmentally friendly, but also allows for the simultaneous extraction of highly active alcohol dehydrogenase and high-purity heparin. This invention provides new ideas for related fields such as the food and pharmaceutical industries.

Description

Technical Field

[0001] This invention relates to the field of chemical manufacturing technology, and in particular to a two-phase aqueous extraction apparatus and a method for simultaneously extracting alcohol dehydrogenase and heparin from pig liver. Background Technology

[0002] Pig liver is a nutritious food with certain medicinal value. Currently, there are numerous patent applications related to pig liver, mainly concerning the preparation and application of pig liver extracts, pig liver health foods and their preparation methods, the screening and application of functional peptides from pig liver, and pig liver feed and its preparation methods.

[0003] Alcohol dehydrogenase, an alcohol-metabolizing enzyme, is commonly found in the livers of mammals. It inhibits the absorption of ethanol in the human body, reducing its damage to the digestive, hepatic, and nervous systems. Alcohol dehydrogenase has broad application prospects in industry, biology, and medicine. As a major by-product of pigs, pig liver is widely available and inexpensive. Given its genetic and physiological characteristics are very similar to those of humans, extracting alcohol dehydrogenase from pig liver to increase its added value and achieve sustainable development is of great significance to the meat processing industry. Traditional alcohol dehydrogenase extraction methods suffer from problems such as cumbersome operation, low extraction efficiency, and unstable processes. Furthermore, the use of organic solvents in these methods poses environmental hazards.

[0004] Heparin is a highly sulfated glycosaminoglycan with wide applications in medicine and healthcare. Currently, the commonly used sources for heparin extraction are porcine intestinal mucosa or bovine lung tissue. Due to limitations of these sources and ethical considerations, the supply of heparin is limited. Porcine liver, as a potential alternative source, contains a considerable amount of heparin. Traditional methods for extracting heparin from porcine liver mainly involve enzymatic hydrolysis and ethanol precipitation. However, this method suffers from problems such as long extraction time, low yield, and low purity, and also consumes significant environmental and resource resources during production, which does not meet the needs of modern industrial production. Therefore, it is necessary to develop an efficient extraction method to improve the yield and purity of heparin, reduce production costs, and achieve sustainable development.

[0005] Aqueous two-phase extraction (APE) technology utilizes simple separation equipment and operates under mild conditions, yielding high product recovery rates and purity, making it suitable for large-scale industrial production. Currently, APE is widely used for the separation and purification of biological products such as proteins, enzymes, and nucleic acids. This method relies on the selective partitioning of substances between two phases. When a substance enters the aqueous two-phase system, the size of the biomolecules, their charge interactions, and the presence of various forces (such as hydrophobic bonds, hydrogen bonds, and ionic bonds), along with environmental influences, result in different concentrations in the upper and lower phases, thus extracting the target protease. This method provides a biocompatible environment for biomolecules, offers continuous operation, and is easily scalable for production. However, there are currently no reports of using APE to extract alcohol dehydrogenase. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies and achieve comprehensive and sustainable utilization of pig liver, this invention aims to provide a two-phase aqueous extraction device and a method for simultaneously extracting alcohol dehydrogenase and heparin from pig liver.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] On the one hand, the present invention provides a two-phase aqueous extraction device, comprising two storage cylinders (1);

[0009] The two storage cylinders (1) are each equipped with a rotating shaft.

[0010] The rotating shaft is fixedly equipped with a drive wheel;

[0011] A first driven wheel is mounted on the left side of the driving wheel;

[0012] A first horizontal shaft is fixedly installed at the center of the first driven wheel;

[0013] The first horizontal axis is rotatably mounted on the inner wall of the storage cylinder;

[0014] The outer wall of the first horizontal shaft is uniformly provided with first stirring rods;

[0015] A second driven wheel is engaged on the right side of the driving wheel;

[0016] A second horizontal shaft is fixedly installed at the center of the second driven wheel;

[0017] The second horizontal axis is rotatably mounted on the inner wall of the storage cylinder;

[0018] The outer wall of the second horizontal shaft is uniformly provided with second stirring rods.

[0019] Furthermore, an extraction tank (13) is connected through to the bottom of the storage cylinder (1);

[0020] The extraction tank (13) is equipped with a discharge valve (8);

[0021] The top of the extraction tank (13) extends to the outside and is fixedly connected to the output shaft (4) of the drive motor (3);

[0022] The drive motor (3) is fixedly installed between the first storage cylinder (1) and the second storage cylinder (1);

[0023] Both storage cylinders (1) are connected to the extraction tank (13) through the feed pipe (2);

[0024] The side wall of the extraction tank (13) is provided with a circulation pipe (9), an extract discharge pipe (12), and an organic phase discharge pipe (14);

[0025] Valves are installed inside the circulation pipe (9), the extract discharge pipe (12), and the organic phase discharge pipe (14);

[0026] The other end of the extract discharge pipe (12) is located at the bottom of the extraction tank (13) to separate the upper phase liquid and the lower phase liquid after extraction and stratification.

[0027] The drive motor (3) includes a reel, a shaft, a motor, and a support base;

[0028] The support base is fixedly installed on the outer wall of the extraction tank (13);

[0029] The drive motor (3) is fixedly installed on one side of the support base;

[0030] One end of the rotating shaft is connected to the output end of the motor, and the other end is connected to the reel.

[0031] Furthermore, the feed pipe (2) is composed of a rotating pipe, a rotary joint, a control valve, and a stirring assembly (5);

[0032] The upper part of the rotating tube is equipped with a rotary joint and a control valve;

[0033] The bottom end of the rotating tube extends into the interior of the mixing cylinder and is uniformly provided with stirring blades on its outer wall.

[0034] Furthermore, the discharge pipe (8) is equipped with a back suction pipe connected to the bottom of the extraction tank (13) for absorbing the lower phase water liquid without absorbing the upper phase oil liquid;

[0035] The stirring assembly (5) consists of a driving pulley, a synchronous belt, a driven pulley, a premixing shaft (10), and a premixing rod (6);

[0036] The top of the stirring assembly (5) is provided with a drive pulley;

[0037] The driving pulley is connected to the driven pulley via a synchronous belt;

[0038] A premixing shaft (10) is fixedly installed at the bottom of the driven pulley;

[0039] The premixing shaft is provided with a premixing rod (6) on its outer wall.

[0040] Secondly, the present invention provides a method for simultaneously extracting alcohol dehydrogenase and heparin from pig liver, using the above-mentioned aqueous two-phase extraction apparatus, comprising the following steps:

[0041] (1) Remove the connective tissue and fat from the pig liver, weigh the remaining liver, cut it into pieces, add it to the prepared Tris-HCl buffer according to the corresponding ratio, homogenize it, let it stand at 4℃ for 2 hours, and freeze and centrifuge for 30 minutes. The resulting supernatant is the crude enzyme solution.

[0042] (2) Add (NH4)2SO4 solid to the crude enzyme solution obtained in step (1) to a certain saturation, and salt out at 4℃ for 2 hours, and collect the supernatant; continue to add (NH4)2SO4 solid to the supernatant to a certain saturation, and salt out at 4℃ for 2 hours, and collect the lower precipitate; dissolve the precipitate in the prepared Tris-HCl buffer to make the solution completely dissolved, stable and homogeneous, and dialyze overnight at 4℃ to obtain the initial enzyme solution;

[0043] (3) Mix a certain proportion of polyethylene glycol (PEG) and ammonium sulfate (NH4)2SO4, add a certain amount of deionized water, shake and mix evenly to form an upper and lower aqueous phase system;

[0044] (4) Add the initial enzyme solution from step two to the aqueous two-phase system from step three, shake and mix for a timed period, let stand for 30 minutes, and separate the upper and lower liquid layers.

[0045] (5) Separation of alcohol dehydrogenase: Transfer the upper liquid from step (4) to a storage tank and repeat step four to extract and separate the lower liquid, which is the alcohol dehydrogenase solution.

[0046] (6) Collect the lower layer liquid from step (5), remove cell debris, impurities, etc. by resin chromatography, and obtain heparin by elution.

[0047] Furthermore, the pH range of the Tris-HCl is 7.0 to 8.5, the ratio of pig liver homogenate to buffer is 1:4 to 1:6, and the refrigerated centrifuge speed is 10,000 r / min to 12,000 r / min.

[0048] Furthermore, (NH4)2SO4 solid was added for the first time to a certain degree of saturation for salting out, with a saturation range of 20% to 40%. The saturation range of (NH4)2SO4 solid was added for the second time to a degree of 60% to 80%. The overnight dialysis time was 24 to 48 hours.

[0049] Furthermore, it includes the following steps:

[0050] (1) Prepare a PEG 6000 solution with a mass fraction of 40% and a (NH4)2SO4 solution with a mass fraction of 20%. Dilute the PEG 6000 solution with a mass fraction of 40% with distilled water to obtain PEG 6000 solutions with different mass fractions.

[0051] (2) Add 20% (NH4)2SO4 solution dropwise to a test tube. Vortex the test tube after each drop of (NH4)2SO4 solution is added. When turbidity appears in the solution system, determine that point as the turbidity point of the aqueous two-phase phase diagram and record the volume of (NH4)2SO4 solution at this point. Calculate the mass fractions of PEG 6 000 solution and (NH4)2SO4 solution at that point. Repeat the recording of the turbidity point multiple times and plot the aqueous two-phase extraction phase diagram with the mass fraction of PEG 6 000 as the ordinate and the mass fraction of (NH4)2SO4 solution as the abscissa.

[0052] (4) Calculate the mass of each phase in the aqueous two-phase system based on the drawn aqueous two-phase phase diagram. Add PEG 6000 solutions of different mass fractions to a dry centrifuge tube, add (NH4)2SO4 in solid form, mix with a certain mass of crude enzyme solution, mix evenly, let stand for 20 min, centrifuge at 4000 r / min for 5 min to obtain the upper and lower two-phase system;

[0053] (5) Fill the tank with a certain volume of ion exchange resin to meet the minimum requirements for ion exchange. The liquid should be prepared at a temperature of 60-80℃, a pH of 7.5-8.5, and a NaCl content of 0.8-1.6%.

[0054] Compared with the prior art, the beneficial effects of the present invention include:

[0055] The system and extraction method provided by this invention feature high purity and low consumption. The process includes a series of carefully designed steps, constructing a two-phase system to efficiently extract alcohol dehydrogenase and heparin simultaneously from pig liver. The extraction efficiency is high, the process is simple, and the operation is easy to master. This method can be widely applied to the comprehensive utilization of pig liver, achieving not only the extraction and purification of alcohol dehydrogenase but also the simultaneous extraction of heparin, thus realizing sustainable development, saving resources, and protecting the environment. This provides new ideas for related fields such as the food and pharmaceutical industries. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0057] Figure 1 The diagram shows a simplified apparatus for aqueous two-phase extraction according to the present invention, wherein: 1. Storage cylinder; 2. Feed pipe; 3. Drive motor; 4. Output shaft; 5. Stirring assembly; 6. Premixing rod; 7. Stirring blade; 8. Discharge pipe; 9. Circulation pipe; 10. Premixing shaft; 11. Stirring blade; 12. Extraction liquid discharge pipe; 13. Extraction tank; 14. Organic phase discharge pipe; 15. Vertical rod.

[0058] Figure 2 This is a technical route diagram for the simultaneous extraction of alcohol dehydrogenase and heparin from pig liver according to the present invention.

[0059] Figure 3 Phase diagrams of aqueous two-phase systems composed of PEG 6000 and (NH4)2SO4 with different mass fractions according to the present invention;

[0060] Figure 4 The image shows an SDS-PAGE electrophoresis result, where 1 represents ethanol dehydrogenase after aqueous two-phase extraction; and 2 represents ethanol dehydrogenase after ammonium sulfate fractionation precipitation. Detailed Implementation

[0061] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0062] Unless otherwise specified, the raw materials used in the embodiments of this invention were all purchased through commercial channels.

[0063] Example 1

[0064] (1) Remove the connective tissue and fat from the fresh pig liver, weigh out 80g of the remaining liver and cut it into small pieces to obtain pig liver paste. The volume ratio of pig liver paste to Tris-HCl buffer is 1:5. Add it to 400mL of prepared pH 8.0 Tris-HCl buffer, homogenize it with a homogenizer, and extract it by standing at 4℃ for 2h to obtain 350mL of the initial system. After freezing and centrifuging at 4℃ and 12000r / min for 30min, the supernatant obtained is 300mL of crude enzyme solution.

[0065] (2) Calculate by mass fraction, add (NH4)2SO4 solid to 300 mL of crude enzyme solution obtained in step (1) until the saturation is 20%, and salt out at 4℃ for 2 h, and collect the supernatant; continue to add (NH4)2SO4 solid to the supernatant until the saturation is 60%, and salt out at 4℃ for 2 h, and collect the lower precipitate; dissolve the precipitate in Tris-HCl buffer at pH 8.0 to make the solution completely dissolved, stable and homogeneous, and dialyze overnight at 4℃ for 24 h to obtain the initial enzyme solution;

[0066] (3) Preparation of the aqueous two-phase system: 40 mL of 26% polyethylene glycol (PEG) and 2 mL of 9% ammonium sulfate (NH4)2SO4 were mixed, 5 mL of deionized water was added, and the mixture was shaken and mixed evenly to form an upper and lower aqueous two-phase system.

[0067] (4) Add the initial enzyme solution from step (2) to the aqueous two-phase system in step (3), shake and mix for a timed period, let stand for 30 minutes, and separate the upper and lower liquid layers.

[0068] (5) Separation of alcohol dehydrogenase: The upper liquid from step four is transferred to a storage tank, and step four is repeated to extract and separate the lower liquid, which is the alcohol dehydrogenase solution. In the orthogonal experiment, the optimal extraction process conditions were selected as follows: (NH4)2SO4 mass fraction of 9%, PEG 6000 mass fraction of 26%, pH 8.0. Under these conditions, the partition coefficient of the enzyme is 3.67 and the extraction rate is 92.39%.

[0069] (6) Collect the lower layer of liquid from (4), remove cell debris and other impurities by resin chromatography, and obtain heparin by elution.

[0070] Preparation of aqueous two-phase systems:

[0071] (1) Referring to the turbidity titration method, prepare a 40% PEG 6000 solution and a 20% (NH4)2SO4 solution. Dilute the 40% PEG 6000 solution with distilled water to obtain PEG 6000 solutions of different mass fractions. Add the 20% (NH4)2SO4 solution dropwise to a test tube using a pipette. After each drop of (NH4)2SO4 solution is added, vortex the test tube. When turbidity appears in the solution system, determine that point as the turbidity point on the aqueous two-phase phase diagram, and record the volume of (NH4)2SO4 solution at this point. Calculate the mass fractions of the PEG 6000 solution and (NH4)2SO4 solution at that point. Repeat this process multiple times, record the turbidity point, and plot the aqueous two-phase extraction phase diagram with the mass fraction of PEG 6000 as the ordinate and the mass fraction of (NH4)2SO4 solution as the abscissa.

[0072] (2) Calculate the mass of each phase in the aqueous two-phase system based on the drawn aqueous two-phase phase diagram. Add PEG 6000 solutions of different mass fractions to dry centrifuge tubes, add (NH4)2SO4 in solid form, mix with a certain mass of crude enzyme solution, mix evenly, let stand for 20 min, centrifuge at 4000 r / min for 5 min to obtain the upper and lower two-phase system.

[0073] (3) The aqueous two-phase system of the hydrophilic polymer polyethylene glycol and (NH4)2SO4 is caused by polymer incompatibility and salting-out, but the aqueous two-phase system can only be formed when the two substances reach a certain concentration in the aqueous solution. For example Figure 1 The figure shows the phase diagram of a two-phase system composed of PEG 6000 and (NH4)2SO4 with different mass fractions. The turbidity points of the two phases are the points on the curve. The lower part of the curve represents the homogeneous mixed system, and the upper part of the curve represents the two-phase system.

[0074] Excel 2016 and Origin 8.6 software were used for plotting, creating orthogonal experimental factor level table 1, and data analysis. SPSS 20.0 software was used for data processing.

[0075] Table 1. Factor Levels in Orthogonal Experiments

[0076]

[0077] The experimental results and range analysis are shown in Table 2, and the variance analysis is shown in Table 3.

[0078] Table 2 Results and Analysis of Orthogonal Experiments

[0079]

[0080]

[0081] The experimental results shown in Table 2 indicate that the primary and secondary factors affecting the aqueous two-phase extraction efficiency of porcine liver ADH are: PEG6000 mass fraction > pH > (NH4)2SO4 mass fraction. The optimal conditions A3B1C2 were determined, meaning that the extraction rate Ye of porcine liver ADH reached its maximum under the conditions of 9% (NH4)2SO4, 26% PEG 6000, and pH 8.0. Clearly, this combination of conditions is not among the nine orthogonal experimental combinations. Therefore, the experiment was repeated under the optimal experimental combination conditions, and the obtained Ye value was 92.39%. Analysis of the results in Table 2 shows that the result of experiment 7 is 4.0% higher than the result of the optimal experimental combination, but the difference is not significant and is within the error range. It is evident that in experimental combination 2, the amount of PEG 6 000 added is less than that in the optimal experiment, but its extraction rate is above 90%. Therefore, from an economic perspective, experimental conditions 2 can be considered as the optimal experimental conditions, namely (NH4)2SO4 10%, PEG 6 000 22%, and pH 8.0.

[0082] Under these conditions, the partition coefficient of the enzyme was 1.92, and the extraction rate was 90.29%. The method of this invention reduces the steps of DEAE-cellulose protein removal and ultrafiltration, while still obtaining electrophoretically pure ADH. Furthermore, compared to the separation and purification of ADH from pig liver via ion exchange column and gel filtration chromatography, the separation and purification cost is low, making it suitable for large-scale production.

[0083] Table 3. Analysis of Variance Results of Orthogonal Tests

[0084]

[0085] Note: Significant difference (P<0.05); Extremely significant difference (P<0.01)

[0086] As shown in Table 3, among the three factors for extracting ADH from pig liver using the aqueous two-phase method, the mass fraction of PEG 6000 had the most significant effect on the extraction rate (P<0.05).

[0087] The molecular weight and purity of ADH extracted from pig liver were determined by SDS-PAGE. A stacking gel with a mass fraction of 5% and a separating gel with a mass fraction of 12% were prepared. 5 μL of sample was mixed with 15 μL of 4× loading buffer and boiled in a 100℃ water bath for 5 min. The sample and marker were loaded simultaneously. The voltage of the stacking gel was set to 90V. After the bromophenol blue indicator entered the separating gel, the voltage was adjusted to 120V. After electrophoresis, the gel was peeled off, stained, destained, and photographed. The relative molecular weight of ADH in pig liver before and after purification was analyzed based on the bands.

[0088] result Figure 4The results show that, based on the molecular weight and relative migration rate of the marker protein in the figure, the molecular weight of the target protease after aqueous two-phase extraction was calculated to be 130 kDa.

[0089] (4) Heparin extraction: A certain volume of ion exchange resin is filled into the tank to meet the minimum requirements for ion exchange. The feed solution should be prepared at a temperature of 70°C, a pH of 8.5, and a NaCl content of 1.6%.

[0090] To achieve the above-mentioned objective of fully performing aqueous two-phase extraction to separate the upper and lower phases, the present invention provides the following technical solution:

[0091] An extraction device for alcohol dehydrogenase in pig liver is designed, comprising two storage cylinders, each containing a rotating shaft. A driving wheel is fixedly mounted on the rotating shaft, and a first driven wheel is mounted on the left side of the driving wheel. A first horizontal shaft is fixedly mounted at the center of the first driven wheel and rotatably disposed on the inner wall of the storage cylinder. A first stirring rod is evenly disposed on the outer wall of the first horizontal shaft. A second driven wheel is engaged on the right side of the driving wheel, and a second horizontal shaft is fixedly mounted at the center of the second driven wheel. The second horizontal shaft is rotatably disposed on the inner wall of a mixing cylinder, and a second stirring rod is evenly disposed on the outer wall of the second horizontal shaft.

[0092] The bottom of the storage cylinder is connected to a discharge pipe, and a discharge valve is installed on the discharge pipe.

[0093] The top of the vertical shaft extends to the outside of the mixing cylinder and is fixedly connected to the output shaft of the drive motor. The drive motor is fixedly installed between the first material cylinder and the second material cylinder. Both the first material cylinder and the second material cylinder are connected to the extraction tank through the feed pipe.

[0094] The extraction tank is equipped with a circulation pipe, an extract discharge pipe, and an organic phase discharge pipe on its side wall. Each of the circulation pipe, extract discharge pipe, and organic phase discharge pipe is equipped with a valve. The other end of the extract discharge pipe is located at the bottom of the extraction tank, which is used to separate the upper phase liquid and the lower phase liquid after extraction and stratification.

[0095] The drive assembly includes a reel, a shaft, a motor, and a support base. The support base is fixedly installed on the outer wall of the extraction tank, the motor is fixedly installed on one side of the support base, one end of the shaft is connected to the output end of the motor, and the other end is connected to the reel.

[0096] The feed pipe consists of a rotating pipe, a rotary joint, a control valve, and stirring blades. The upper part of the rotating pipe is equipped with a rotary joint and a control valve, and the bottom end of the rotating pipe extends into the interior of the mixing cylinder and is uniformly equipped with stirring blades on its outer wall.

[0097] The discharge pipe is equipped with a back suction pipe connected to the bottom of the extraction tank, which is used to draw in the lower phase water liquid but not the upper phase oil liquid.

[0098] The mixing assembly comprises a driving pulley, a synchronous belt, a driven pulley, a premixing shaft, and a premixing rod. The driving pulley is located at the top of the vertical rod, and the driven pulley is connected to the driven pulley via the synchronous belt. The premixing shaft is fixedly mounted at the bottom of the driven pulley, and the premixing rod is mounted on the outer wall of the premixing shaft. The above description is merely a preferred embodiment of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneous extraction of alcohol dehydrogenase and heparin from pig liver, characterized in that, Includes the following steps: (1) The connective tissue and fat of pig liver were removed, the rest of liver was weighed and cut into pieces, and then added into the prepared Tris-HCl buffer solution according to the corresponding proportion, homogenized, and then extracted at 4-8℃ for 2-3h, and then frozen and centrifuged for 30-40min to obtain the supernatant as the crude enzyme solution. (1) The connective tissue and fat of pig liver were removed, the rest of liver was weighed and cut into pieces, and then added into the prepared Tris-HCl buffer solution according to the corresponding proportion, homogenized, and then extracted at 4-8℃ for 2-3h, and then frozen and centrifuged for 30-40min to obtain the supernatant as the crude enzyme solution. (2) Add (NH4)2SO4 solid to the crude enzyme solution obtained in step (1) until a certain saturation is reached, and perform a first salting-out process at 4-8℃ for 2-3 hours, collecting the supernatant; continue adding (NH4)2SO4 solid to the supernatant until a certain saturation is reached, and perform a second salting-out process at 4-8℃ for 2-3 hours, collecting the lower precipitate; then add the prepared Tris... Dissolve the precipitate in HCl buffer to ensure complete dissolution and a stable, homogeneous solution. Dialyze overnight at 4–8°C to obtain the initial enzyme solution. (3) Mix a certain proportion of polyethylene glycol (PEG) and ammonium sulfate (NH4)2SO4, add a certain amount of deionized water, shake and mix evenly to form an upper and lower aqueous phase system; (4) Add the initial enzyme solution from step (2) to the aqueous two-phase system from step (3), shake and mix for a timed period, let stand for 30 minutes, and separate the upper and lower liquid layers. (5) Separation of alcohol dehydrogenase: Transfer the upper liquid from step (4) to a storage tank and repeat step four to extract and separate the lower liquid, which is the alcohol dehydrogenase solution. (6) Collect the lower layer liquid from step (5), remove cell debris and extraneous proteins by resin chromatography, and obtain heparin by elution. The method for simultaneously extracting alcohol dehydrogenase and heparin from pig liver is performed using an aqueous two-phase extraction device, which includes: Two storage cylinders (1); The two storage cylinders (1) are each equipped with a rotating shaft. The rotating shaft is fixedly equipped with a drive wheel; A first driven wheel is mounted on the left side of the driving wheel; A first horizontal shaft is fixedly installed at the center of the first driven wheel; The first horizontal axis is rotatably mounted on the inner wall of the storage cylinder; The outer wall of the first horizontal shaft is uniformly provided with first stirring rods; A second driven wheel is engaged on the right side of the driving wheel; A second horizontal shaft is fixedly installed at the center of the second driven wheel; The second horizontal axis is rotatably mounted on the inner wall of the storage cylinder; The outer wall of the second horizontal shaft is uniformly provided with second stirring rods.

2. The method for simultaneously extracting alcohol dehydrogenase and heparin from pig liver according to claim 1, characterized in that, The bottom of the storage cylinder (1) is connected to the extraction tank (13); The extraction tank (13) is equipped with a discharge pipe (8); The top of the extraction tank (13) extends to the outside and is fixedly connected to the output shaft (4) of the drive motor (3); The drive motor (3) is fixedly installed between the first storage cylinder (1) and the second storage cylinder (1); Both storage cylinders (1) are connected to the extraction tank (13) through the feed pipe (2); The side wall of the extraction tank (13) is provided with a circulation pipe (9), an extract discharge pipe (12), and an organic phase discharge pipe (14); Valves are installed inside the circulation pipe (9), the extract discharge pipe (12), and the organic phase discharge pipe (14); The other end of the extract discharge pipe (12) is located at the bottom of the extraction tank (13) to separate the upper phase liquid and the lower phase liquid after extraction and stratification. The drive motor (3) includes a reel, a shaft, a motor, and a support base; The support base is fixedly installed on the outer wall of the extraction tank (13); The drive motor (3) is fixedly installed on one side of the support base; One end of the rotating shaft is connected to the output end of the motor, and the other end is connected to the reel.

3. The method for simultaneously extracting alcohol dehydrogenase and heparin from pig liver according to claim 2, characterized in that, The feed pipe (2) consists of a rotating pipe, a rotary joint, a control valve, and a stirring blade (7); The upper part of the rotating tube is equipped with a rotary joint and a control valve; The bottom end of the rotating tube extends into the interior of the mixing cylinder and is uniformly provided with stirring blades on its outer wall.

4. The method for simultaneously extracting alcohol dehydrogenase and heparin from pig liver according to claim 3, characterized in that, The discharge pipe (8) is equipped with an extract discharge pipe connected to the bottom of the extraction tank (13) for absorbing the lower phase water liquid without absorbing the upper phase oil liquid; The mixing assembly (5) consists of a drive pulley, a synchronous belt, a driven pulley, a premixing shaft (10), and a premixing rod (6); The top of the stirring assembly (5) is provided with an active pulley; The driving pulley is connected to the driven pulley via a synchronous belt; A premixing shaft (10) is fixedly installed at the bottom of the driven pulley; The premixing shaft is provided with a premixing rod (6) on its outer wall.

5. The method as described in claim 1, characterized in that, The pH range of Tris-HCl in step (1) is 7.0 to 8.5, the ratio of pig liver homogenate to buffer is 1:4 to 1:6, and the speed of the refrigerated centrifuge is 10000r / min to 12000r / min.

6. The method as described in claim 1, characterized in that, The first addition of (NH4)2SO4 solid to a certain saturation level was used for salting out, with a saturation range of 20% to 40%. The second addition of (NH4)2SO4 solid was used to achieve a saturation range of 60% to 80%, and the overnight dialysis time was 24 to 48 hours.

7. The method according to any one of claims 1 to 6, characterized in that, The preparation method of the aqueous two-phase system described in step (3) includes the following steps: (1) Prepare a 40% PEG6000 solution and a 20% (NH4)2SO4 solution. Dilute the 40% PEG6000 solution with distilled water to obtain PEG6000 solutions with different mass fractions. (2) Add the 20% (NH4)2SO4 solution dropwise to a test tube. Vortex the test tube after each drop of (NH4)2SO4 solution is added. When turbidity appears in the solution system, determine that point as the turbidity point of the aqueous two-phase phase diagram and record the volume of (NH4)2SO4 solution at this point. Calculate the mass fractions of the PEG6000 solution and (NH4)2SO4 solution at that point. Repeat the recording of the turbidity point multiple times and plot the aqueous two-phase extraction phase diagram with the mass fraction of PEG6000 as the ordinate and the mass fraction of (NH4)2SO4 solution as the abscissa. (3) Based on the drawn aqueous phase diagram, calculate the mass of each phase in the aqueous system, add PEG6000 solution with different mass fractions to a dry centrifuge tube, add (NH4)2SO4 in solid form, mix a certain mass of crude enzyme solution, mix evenly, let stand for 20 min, centrifuge at 4000 r / min for 5 min to obtain the upper and lower phase system. (4) Fill the tank with a certain volume of ion exchange resin to meet the minimum requirements for ion exchange. The liquid should be prepared with a temperature of 60-80℃, a pH of 7.5-8.5, and a NaCl content of 0.8-1.6%.

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