CN114317653A - Method for extracting elastin by using bovine heart vessels - Google Patents
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- CN114317653A CN114317653A CN202111593142.XA CN202111593142A CN114317653A CN 114317653 A CN114317653 A CN 114317653A CN 202111593142 A CN202111593142 A CN 202111593142A CN 114317653 A CN114317653 A CN 114317653A
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Separation Using Semi-Permeable Membranes (AREA)
Abstract
The invention discloses a method for extracting elastin by utilizing bovine heart vessels, and relates to the technical field of protein extraction. Firstly, after ground bovine heart vessels are heated by alkali liquor microwaves, protein is extracted by using hydrolytic protease under the assistance of ultrasonic waves, then neutral protease is combined with macroporous resin for adsorption and decoloration, the loss of elastin is reduced, then enzyme is inactivated by infrared heating, and preliminary concentration is realized; and then reducing the microbial content of the elastin by a self-made sterilization film, further concentrating, and finally solidifying the elastin by spray freeze drying, wherein the self-made sterilization film utilizes dodecyl phthalate and dodecyl dimethylamine to modify cellulose and is crosslinked with polyvinylidene fluoride under the assistance of ultrasonic waves, so that the self-made sterilization film is endowed with high-efficiency sterilization and excellent mechanical properties. The method for extracting elastin by using bovine heart vessels has the advantages of high purity, low microorganism content and short time consumption.
Description
Technical Field
The invention relates to the technical field of protein extraction, in particular to a method for extracting elastin by utilizing bovine heart vessels.
Background
Elastin is insoluble and highly cross-linked macromolecular fibrous protein in extracellular matrix, can be combined with microfibril in vivo to form elastic fiber, has the main function of passive stretching, and endows tissues and organs with stretching and reversible deformability, so that the elastin exists in organs and tissues based on elasticity, such as ligaments, arteries, lungs, skin and the like in a large amount.
With the increasing demand of the market for elastin, the existing extraction process has certain defects; after the elastin is extracted by enzymolysis, cotton wool is often mixed in the solution by using cotton cake for decolorization, so that the quality and the purity of the elastin are influenced, a large amount of impurities are introduced, and a small amount of cotton wool remains in the elastin even after subsequent filtration, so that the actual application of the elastin is influenced; in addition, in the process of sterilization treatment, the biological activity of elastin is influenced to a certain extent, the molecular chain of the elastin can be damaged, and after multiple times of sterilization, although the content of microorganisms is reduced, elastin is seriously lost, so that the actual weight is greatly reduced. Therefore, a more efficient elastin extraction process is needed.
Disclosure of Invention
The invention aims to provide a method for extracting elastin by utilizing bovine heart vessels, which aims to solve the problems in the prior art.
In order to solve the technical problems, the invention provides the following technical scheme: a method for extracting elastin by using bovine heart vessels is characterized by mainly comprising the following preparation steps:
(1) placing the bovine heart vessels and purified water with the mass of 3-5 times of the bovine heart vessels into a colloid mill pulverizer, and pulverizing for 3-5 min to obtain slurry;
(2) placing the slurry in a glass container, adding 5% by mass of sodium hydroxide until the pH of the solution is 7.0-7.5, placing the solution in a microwave oven with the microwave power of 600-700W, heating for 50-70 s, and cooling to room temperature to obtain a heating extracting solution;
(3) putting the heated extracting solution into an ultrasonic cleaning machine, adding hydrolysis protease with the ultrasonic power of 140-160W, wherein the mass of the hydrolysis protease is 0.03 times that of bovine heart vessels, and extracting for 40-60 min to obtain an enzymolysis extracting solution;
(4) placing the enzymolysis extracting solution into a centrifuge, centrifuging at 4000-4500 rpm for 10-15 min, placing into a 50 ℃ constant temperature water bath kettle, adding neutral protease with the mass of 0.01 time of that of bovine heart vessels, performing enzymolysis for 9-11 min, adding macroporous resin with the mass of 0.1 time of that of the bovine heart vessels, adsorbing for 40-50 min, and filtering to obtain a supernatant;
(5) placing the supernatant in an infrared generator, wherein the infrared energy intensity is 3-5 kW/m2Inactivating enzyme for 6-8 min, and adding 5% by mass of hydrochloric acid until the pH of the solution is 4.8-5.0; then placing the mixture in a centrifuge, and centrifuging the mixture at 6000-6200 rpm for 13-15 min to obtain elastin;
(6) sterilizing the elastin by a self-made sterilization membrane;
(7) and (4) carrying out spray freeze drying on the sterilized elastin to finish the elastin extraction by utilizing the bovine heart vessels.
Further, the linear speed of a rotor in the colloid mill pulverizer in the step (1) is 20-30 m/s.
Further, the infrared generator in the step (5) is preheated for 20min before being used, and the temperature reaches 180 ℃.
Further, the preparation method of the self-made sterilization membrane in the step (6) comprises the following steps:
a. dissolving cellulose in deionized water with the mass 40-45 times of that of the cellulose, ultrasonically dissolving for 3-5 min at 200-250W, heating to 80-100 ℃, adding dodecyl phthalate with the mass 1.2-1.5 times of that of the cellulose, stirring for 20-30 min at 200-300 rpm, reacting for 50-60 min at 200-300W ultrasonic power, cooling to room temperature, and rotationally steaming for 3-5 h at 300rpm and 80 ℃ to obtain arylated cellulose;
b. adding arylated cellulose into a three-necked flask, stirring at 50rpm, adding hydrochloric acid with the mass fraction of 65% and paraformaldehyde with the mass fraction of 0.35-0.4 time of the arylated cellulose, wherein the hydrochloric acid is 7.5-8 times of the mass of the arylated cellulose, the paraformaldehyde is 0.35-0.4 time of the mass of the arylated cellulose, the temperature is controlled at 15-25 ℃, phosphorus oxychloride with the mass fraction of 0.4-0.5 time of the arylated cellulose is dropwise added at the speed of 0.5-0.7 mL/min, reacting for 16-20 hours at 300-400 rpm, performing suction filtration, washing with deionized water and a sodium bicarbonate aqueous solution for 7-10 times in sequence, wherein the mass ratio of sodium bicarbonate to deionized water in the sodium bicarbonate aqueous solution is 1:32, washing with distilled water until the pH of the solution is 6-7, 60 ℃ and 0.02MPa, and performing vacuum drying for 2-3 hours to obtain chlorinated cellulose;
c. adding chlorinated cellulose and ethyl acetate which is 3-3.5 times of the mass of the chlorinated cellulose into a reaction bottle, stirring for dissolving, adding dodecyl dimethylamine which is 0.6-0.7 times of the mass of the chlorinated cellulose under cooling in ice-water bath at 0-2 ℃, reacting for 20-24 h at room temperature, and filtering to obtain modified cellulose;
d. placing polyvinylidene fluoride in a vacuum oven with the temperature of 100 ℃ and the pressure of 0.08MPa for drying for 20-24 h, adding triethyl phosphate with the mass being 2-3 times that of the polyvinylidene fluoride, stirring for 4-5 h at 200-300 rpm, placing in an outer spinning feed liquid tank, and standing and defoaming for 12-14 h; adding modified cellulose, dimethylformamide, acetone and glycerol into the inner spinning feed liquid tank according to the mass ratio of 1:1.45:0.88: 3; stirring at 200-300 rpm for 2-4 h, standing for defoaming for 8-10 h, and co-extruding to obtain a hollow fiber membrane;
e. and (3) treating the hollow fiber membrane for 5-10 min under ultrasonic irradiation with power of 300-400W at 145-155 ℃, standing for 2-3 h at room temperature, and thus obtaining the antibacterial membrane.
Furthermore, in the coextrusion process in the step d, the speed of the spinning solution of the outer layer is 0.2mL/min, the speed of the spinning solution of the inner layer is 0.8mL/min, the speed of the core solution is 0.3mL/min, the temperature of a spray head is 75 ℃, the condensation temperature is 15 ℃, and the condensation source is tap water; the core liquid is formed by mixing methyl pyrrolidone and deionized water according to the volume ratio of 95: 5.
Further, the cold soaking temperature of the spray freeze drying in the step (7) is-90 to-80 ℃, and the feeding amount is 10 to 15 mL/min.
Compared with the prior art, the invention has the following beneficial effects:
the method utilizes the bovine heart vessels to extract the elastin through the steps of microwave heating, ultrasonic enzymolysis, adsorption decoloration, infrared enzyme inactivation, sterilization, spray freeze drying and the like, and has the advantages of simple operation, high purity of the extracted elastin and low content of microorganisms.
Firstly, heating the bovine heart vessels by alkali liquor microwave to fully swell the elastic fibers, thereby being beneficial to extracting elastin subsequently; then, under the assistance of ultrasonic waves, protein is extracted efficiently and quickly by using hydrolytic protease; the adsorption decoloration is carried out by utilizing the restriction enzyme hydrolysis of neutral protease and combining with macroporous resin, the effect is obvious, and the loss rate of elastin is low; then infrared heating is carried out to inactivate enzyme, the time is short, the efficiency is high, partial dehydration can be achieved, and preliminary concentration is realized; then reducing the microbial content of elastin by a self-made sterilization membrane, and further concentrating; the elastin is solidified by spray freeze drying to form porous particles, which have interconnected pore grid structure, large specific surface area and good rehydration property.
Secondly, co-extruding the self-made sterilization membrane by adopting modified cellulose as an inner layer and polyvinylidene fluoride as an outer layer to prepare a double-layer asymmetric hollow fiber membrane; the modified cellulose is prepared from cellulose, dodecyl phthalate and dodecyl dimethylamine; under the action of ultrasonic wave, carboxyl of dodecyl phthalate reacts with hydroxyl of cellulose; the cellulose structure becomes loose by ultrasonic waves, so that the dodecyl phthalate intercalation reaction is facilitated, and the reaction rate is accelerated; then, the dodecyl phthalate is chlorinated and reacts with tertiary amino of dodecyl dimethylamine to form an antibacterial group quaternary ammonium salt, so that the modified cellulose has a sterilization effect and microorganisms in elastin are reduced; the dodecyl phthalate and the dodecyl dimethylamine can excite the activity of antibacterial groups, improve the bacteriostatic effect of a self-made antibacterial film, effectively remove bacteria in elastin, and improve the hydrophobicity of modified cellulose by introducing a plurality of hydrophobic groups, thereby being beneficial to concentrating elastin; after the self-made sterilization membrane is prepared into a membrane, a polyvinylidene fluoride molecular chain penetrates through the modified cellulose layers under the action of ultrasonic irradiation, and the modified cellulose structure is polymerized again due to the secondary ultrasonic irradiation of the modified cellulose, so that the modified cellulose and the modified cellulose are tightly crosslinked, the membrane layering is reduced, and the mechanical property of the self-made sterilization membrane is improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to 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.
To illustrate the method of the present invention more clearly, the following examples are given, in which the following test methods for each index of elastin were extracted:
purity: the elastin extracted from the example and the comparative example with the same mass is used for determining purity by mass spectrometry and gel separation chromatography.
The extraction rate is as follows: the elastin extracted from the same mass example and comparative example is measured by Kjeldahl method to calculate the elastin extraction rate. The elastin extraction rate ═ mass of elastin/mass of bovine heart vessel x 100%.
The content of microorganisms: the elastin extracted from the same mass example and comparative example is used for determining the microbial content, and the total colony content, coliform group content and mould content are determined respectively by reference to GB 4789.2, GB 4789.3 and GB 4789.2.
Example 1
A method for extracting elastin by using bovine heart vessels mainly comprises the following preparation steps:
(1) placing bovine heart vessel and purified water with the weight 3 times of that of the bovine heart vessel in a colloid mill pulverizer, and pulverizing for 3min to obtain slurry;
(2) placing the slurry in a glass container, adding 5% by mass of sodium hydroxide until the pH of the solution is 7.0, placing the solution in a microwave oven with the microwave power of 600W, heating for 70s, and cooling to room temperature to obtain a heated extracting solution;
(3) placing the heated extract in an ultrasonic cleaning machine with ultrasonic power of 140W, adding 0.03 times of hydrolytic protease of bovine heart vessel, and extracting for 40min to obtain enzymolysis extract;
(4) placing the enzymolysis extractive solution in a centrifuge, centrifuging at 4000rpm for 15min, placing in a 50 deg.C constant temperature water bath kettle, adding neutral protease 0.01 times the mass of bovine heart vessel, performing enzymolysis for 9min, adding macroporous resin 0.1 times the mass of bovine heart vessel, adsorbing for 40min, and filtering to obtain supernatant;
(5) placing the supernatant in an infrared generator with infrared energy intensity of 3kW/m2Inactivating enzyme for 8min, and adding 5% hydrochloric acid until pH is 4.8; then placing the mixture in a centrifuge, and centrifuging the mixture at 6000rpm for 15min to obtain elastin;
(6) sterilizing the elastin by a self-made sterilization membrane;
(7) and (4) carrying out spray freeze drying on the sterilized elastin to finish the elastin extraction by utilizing the bovine heart vessels.
Further, the linear speed of a rotor in the colloid mill pulverizer in the step (1) is 20 m/s.
Further, the infrared generator in the step (5) is preheated for 20min before being used, and the temperature reaches 180 ℃.
Further, the preparation method of the self-made sterilization membrane in the step (6) comprises the following steps:
a. dissolving cellulose in deionized water 40 times of the mass of the cellulose, ultrasonically dissolving for 3min at 200W, heating to 80 ℃, adding dodecyl phthalate 1.2 times of the mass of the cellulose, stirring for 30min at 200rpm, reacting for 60min at 200W ultrasonic power, cooling to room temperature, and rotatably steaming for 3h at 300rpm and 80 ℃ to obtain arylated cellulose;
b. adding arylated cellulose into a three-neck flask, stirring at the speed of 50rpm, adding hydrochloric acid with the mass fraction of 65% and paraformaldehyde with the mass fraction of 0.35 times of the mass of the arylated cellulose, wherein the hydrochloric acid is 7.5 times of the mass of the arylated cellulose, adding phosphorus oxychloride with the mass fraction of 0.4 times of the mass of the arylated cellulose dropwise at the speed of 0.5mL/min, reacting for 20 hours at 300rpm, performing suction filtration, washing for 7 times by using deionized water and a sodium bicarbonate aqueous solution in sequence, wherein the mass ratio of sodium bicarbonate to deionized water in the sodium bicarbonate aqueous solution is 1:32, washing with distilled water until the pH of the solution is 6, 60 ℃ and 0.02MPa, and performing vacuum drying for 2 hours to obtain chlorinated cellulose;
c. adding chlorinated cellulose and ethyl acetate with the mass 3 times of that of the chlorinated cellulose into a reaction bottle, stirring and dissolving, adding dodecyl dimethylamine with the mass 0.6 time of that of the chlorinated cellulose under the cooling of ice-water bath at 0 ℃, reacting for 20 hours at room temperature, and filtering to obtain modified cellulose;
d. placing polyvinylidene fluoride in a vacuum oven with the temperature of 100 ℃ and the pressure of 0.08MPa for drying for 20h, adding triethyl phosphate with the mass 2 times that of the polyvinylidene fluoride, stirring for 4h at 200rpm, placing in an outer layer spinning feed liquid tank, standing and defoaming for 12 h; adding modified cellulose, dimethylformamide, acetone and glycerol into the inner spinning feed liquid tank according to the mass ratio of 1:1.45:0.88: 3; stirring at 200rpm for 4h, standing for defoaming for 8h, and co-extruding to obtain a hollow fiber membrane;
e. treating the hollow fiber membrane under ultrasonic irradiation with power of 300W for 10min at 145 ℃, and standing for 2h at room temperature to obtain the antibacterial membrane.
Furthermore, in the coextrusion process in the step d, the speed of the spinning solution of the outer layer is 0.2mL/min, the speed of the spinning solution of the inner layer is 0.8mL/min, the speed of the core solution is 0.3mL/min, the temperature of a spray head is 75 ℃, the condensation temperature is 15 ℃, and the condensation source is tap water; the core liquid is formed by mixing methyl pyrrolidone and deionized water according to the volume ratio of 95: 5.
Further, the cold soaking temperature of the spray freeze drying in the step (7) is-90 ℃, and the feeding amount is 10 mL/min.
Example 2
A method for extracting elastin by using bovine heart vessels mainly comprises the following preparation steps:
(1) placing cattle heart vessel and purified water with 5 times of the weight of the cattle heart vessel in a colloid mill pulverizer, pulverizing for 5min to obtain slurry;
(2) placing the slurry in a glass container, adding 5% by mass of sodium hydroxide until the pH of the solution is 7.5, placing the solution in a microwave oven with the microwave power of 700W, heating for 50s, and cooling to room temperature to obtain a heated extracting solution;
(3) placing the heated extract in an ultrasonic cleaning machine with ultrasonic power of 160W, adding 0.03 times of hydrolytic protease of bovine heart vessel, and extracting for 40min to obtain enzymolysis extract;
(4) placing the enzymolysis extractive solution in a centrifuge, centrifuging at 4500rpm for 10min, placing in a 50 deg.C constant temperature water bath, adding neutral protease 0.01 times the mass of heart vessel of cattle, performing enzymolysis for 11min, adding macroporous resin 0.1 times the mass of heart vessel of cattle, adsorbing for 50min, and filtering to obtain supernatant;
(5) placing the supernatant in an infrared generator with infrared energy intensity of 5kW/m2Inactivating enzyme for 6min, and adding 5% hydrochloric acid until pH of the solution is 5.0; then placing the mixture in a centrifuge, and centrifuging the mixture for 13min at 6200rpm to obtain elastin;
(6) sterilizing the elastin by a self-made sterilization membrane;
(7) and (4) carrying out spray freeze drying on the sterilized elastin to finish the elastin extraction by utilizing the bovine heart vessels.
Further, the linear speed of a rotor in the colloid mill pulverizer in the step (1) is 30 m/s.
Further, the infrared generator in the step (5) is preheated for 20min before being used, and the temperature reaches 180 ℃.
Further, the preparation method of the self-made sterilization membrane in the step (6) comprises the following steps:
a. dissolving cellulose in deionized water 45 times of the mass of the cellulose, ultrasonically dissolving for 3min at 250W, heating to 100 ℃, adding dodecyl phthalate 1.5 times of the mass of the cellulose, stirring for 20min at 300rpm, reacting for 50min at 300W ultrasonic power, cooling to room temperature, and rotationally steaming for 5h at 300rpm and 80 ℃ to obtain arylated cellulose;
b. adding arylated cellulose into a three-neck flask, stirring at the speed of 50rpm, adding hydrochloric acid with the mass fraction of 65% and paraformaldehyde with the mass fraction of 0.4 time of the arylated cellulose, the mass fraction of which is 8 times of the mass of the arylated cellulose, dropwise adding phosphorus oxychloride with the mass fraction of 0.5 time of the mass of the arylated cellulose at the speed of 0.7mL/min, reacting for 16 hours at the speed of 400rpm, performing suction filtration, washing for 10 times by using deionized water and a sodium bicarbonate aqueous solution in sequence, wherein the mass ratio of sodium bicarbonate to deionized water in the sodium bicarbonate aqueous solution is 1:32, washing by using distilled water until the pH of the solution is 7, 60 ℃ and 0.02MPa, and performing vacuum drying for 3 hours to obtain chlorinated cellulose;
c. adding chlorinated cellulose and ethyl acetate with the mass 3.5 times of that of the chlorinated cellulose into a reaction bottle, stirring and dissolving, adding dodecyl dimethylamine with the mass 0.7 time of that of the chlorinated cellulose under the cooling of ice-water bath at the temperature of 2 ℃, reacting for 24 hours at room temperature, and filtering to obtain modified cellulose;
d. placing polyvinylidene fluoride in a vacuum oven with the temperature of 100 ℃ and the pressure of 0.08MPa for drying for 24h, adding triethyl phosphate with the mass of 3 times that of the polyvinylidene fluoride, stirring for 5h at 300rpm, placing in an outer-layer spinning feed liquid tank, standing and defoaming for 14 h; adding modified cellulose, dimethylformamide, acetone and glycerol into the inner spinning feed liquid tank according to the mass ratio of 1:1.45:0.88: 3; stirring for 2h at 300rpm, standing for defoaming for 10h, and co-extruding to obtain a hollow fiber membrane;
e. treating the hollow fiber membrane under 400W ultrasonic irradiation for 5min at 155 ℃, standing for 3h at room temperature, and thus obtaining the sterilization membrane.
Furthermore, in the coextrusion process in the step d, the speed of the spinning solution of the outer layer is 0.2mL/min, the speed of the spinning solution of the inner layer is 0.8mL/min, the speed of the core solution is 0.3mL/min, the temperature of a spray head is 75 ℃, the condensation temperature is 15 ℃, and the condensation source is tap water; the core liquid is formed by mixing methyl pyrrolidone and deionized water according to the volume ratio of 95: 5.
Further, the cold soaking temperature of the spray freeze drying in the step (7) is-80 ℃, and the feeding amount is 15 mL/min.
Comparative example 1
A method for extracting elastin by using bovine heart vessels mainly comprises the following preparation steps:
(1) placing the bovine heart vessels and purified water with the mass of 4.2 times of the bovine heart vessels in a colloid mill pulverizer, and pulverizing for 4min to obtain slurry;
(2) placing the slurry in a glass container, adding 5% by mass of sodium hydroxide until the pH of the solution is 7.2, placing the slurry in a microwave oven with the microwave power of 630W, heating for 60s, and cooling to room temperature to obtain a heated extracting solution;
(3) adding 0.03 times of hydrolytic protease of bovine heart vessel mass into the heated extracting solution, and carrying out enzymolysis for 8h to obtain an enzymolysis extracting solution;
(4) placing the enzymolysis extractive solution in a centrifuge, centrifuging at 4350rpm for 13min, placing in 50 deg.C constant temperature water bath, adding neutral protease 0.01 times the mass of heart vessel of cattle, performing enzymolysis for 10min, adding macroporous resin 0.1 times the mass of heart vessel of cattle, adsorbing for 43min, and filtering to obtain supernatant;
(5) placing the supernatant in an infrared generator with infrared energy intensity of 4kW/m2Inactivating enzyme for 7min, and adding 5% hydrochloric acid until pH is 4.9; then placing the mixture in a centrifuge, and centrifuging the mixture for 14min at 6150rpm to obtain elastin;
(6) sterilizing the elastin by a self-made sterilization membrane;
(7) and (4) carrying out spray freeze drying on the sterilized elastin to finish the elastin extraction by utilizing the bovine heart vessels.
Further, the linear speed of a rotor in the colloid mill pulverizer in the step (1) is 25 m/s.
Further, the infrared generator in the step (5) is preheated for 20min before being used, and the temperature reaches 180 ℃.
Further, the preparation method of the self-made sterilization membrane in the step (6) comprises the following steps:
a. dissolving cellulose in deionized water with the mass of 43.2 times of that of the cellulose, ultrasonically dissolving the cellulose for 4min at 230W, heating to 90 ℃, adding dodecyl phthalate with the mass of 1.36 times of that of the cellulose, stirring the mixture for 25min at 260rpm, reacting the mixture for 53min at 276W ultrasonic power, cooling to room temperature, and rotationally steaming the mixture for 4h at 300rpm and 80 ℃ to obtain arylated cellulose;
b. adding arylated cellulose into a three-neck flask, stirring at the speed of 50rpm, adding hydrochloric acid with the mass fraction of 65% and paraformaldehyde with the mass fraction of 0.38 times of the mass of the arylated cellulose, wherein the hydrochloric acid is 7.84 times of the mass of the arylated cellulose, the paraformaldehyde is 0.38 times of the mass of the arylated cellulose, the temperature is controlled at 20 ℃, phosphorus oxychloride with the mass fraction of 0.45 time of the mass of the arylated cellulose is dropwise added at the speed of 0.65mL/min, reacting for 18 hours at 330rpm, performing suction filtration, washing for 8 times by using deionized water and a sodium bicarbonate aqueous solution in sequence, wherein the mass ratio of sodium bicarbonate to deionized water is 1:32, washing with distilled water until the pH of the solution is 6.5, 60 ℃ and 0.02MPa, and performing vacuum drying for 2.5 hours to obtain cellulose chloride;
c. adding chlorinated cellulose and ethyl acetate with the mass 3.27 times of that of the chlorinated cellulose into a reaction bottle, stirring and dissolving, adding dodecyl dimethylamine with the mass 0.67 time of that of the chlorinated cellulose under the cooling of ice-water bath at the temperature of 1 ℃, reacting for 22 hours at room temperature, and filtering to obtain modified cellulose;
d. placing polyvinylidene fluoride in a vacuum oven with the temperature of 100 ℃ and the pressure of 0.08MPa for drying for 23h, adding triethyl phosphate with the mass of 2.45 times that of the polyvinylidene fluoride, stirring for 4.2h at 240rpm, placing in an outer-layer spinning feed liquid tank, standing and defoaming for 13 h; adding modified cellulose, dimethylformamide, acetone and glycerol into the inner spinning feed liquid tank according to the mass ratio of 1:1.45:0.88: 3; stirring for 3.5h at 260rpm, standing for defoaming for 9h, and co-extruding to obtain a hollow fiber membrane;
e. the hollow fiber membrane is treated for 7min under the ultrasonic irradiation with the power of 340W at the temperature of 150 ℃, and then is kept stand for 2.5h at room temperature, thus obtaining the sterilization membrane.
Furthermore, in the coextrusion process in the step d, the speed of the spinning solution of the outer layer is 0.2mL/min, the speed of the spinning solution of the inner layer is 0.8mL/min, the speed of the core solution is 0.3mL/min, the temperature of a spray head is 75 ℃, the condensation temperature is 15 ℃, and the condensation source is tap water; the core liquid is formed by mixing methyl pyrrolidone and deionized water according to the volume ratio of 95: 5.
Further, the cold soaking temperature of the spray freeze drying in the step (7) is-85 ℃, and the feeding amount is 13 mL/min.
Comparative example 2
A method for extracting elastin by using bovine heart vessels mainly comprises the following preparation steps:
(1) placing the bovine heart vessels and purified water with the mass of 4.2 times of the bovine heart vessels in a colloid mill pulverizer, and pulverizing for 4min to obtain slurry;
(2) placing the slurry in a glass container, adding 5% by mass of sodium hydroxide until the pH of the solution is 7.2, placing the slurry in a microwave oven with the microwave power of 630W, heating for 60s, and cooling to room temperature to obtain a heated extracting solution;
(3) placing the heated extract in an ultrasonic cleaning machine with ultrasonic power of 150W, adding 0.03 times of hydrolytic protease of bovine heart vessel, and extracting for 50min to obtain enzymolysis extract;
(4) placing the enzymolysis extractive solution in a centrifuge, centrifuging at 4350rpm for 13min, and placing in an infrared generator with infrared energy intensity of 4kW/m2Inactivating enzyme for 7min, and adding 5% hydrochloric acid until pH is 4.9 to obtain elastin;
(5) standing elastin for 4h, and taking supernatant; filtering the supernatant with cotton cake filter, adding active carbon 0.0001 times the weight of the supernatant, heating to 80 deg.C, maintaining the temperature for 90min, filtering with plate-and-frame filter, and decolorizing;
(6) sterilizing the decolorized elastin by a self-made sterilization membrane;
(7) and (4) carrying out spray freeze drying on the sterilized elastin to finish the elastin extraction by utilizing the bovine heart vessels.
Further, the linear speed of a rotor in the colloid mill pulverizer in the step (1) is 25 m/s.
Further, the infrared generator in the step (4) is preheated for 20min before being used, and the temperature reaches 180 ℃.
Further, the preparation method of the self-made sterilization membrane in the step (6) comprises the following steps:
a. dissolving cellulose in deionized water with the mass of 43.2 times of that of the cellulose, ultrasonically dissolving the cellulose for 4min at 230W, heating to 90 ℃, adding dodecyl phthalate with the mass of 1.36 times of that of the cellulose, stirring the mixture for 25min at 260rpm, reacting the mixture for 53min at 276W ultrasonic power, cooling to room temperature, and rotationally steaming the mixture for 4h at 300rpm and 80 ℃ to obtain arylated cellulose;
b. adding arylated cellulose into a three-neck flask, stirring at the speed of 50rpm, adding hydrochloric acid with the mass fraction of 65% and paraformaldehyde with the mass fraction of 0.38 times of the mass of the arylated cellulose, wherein the hydrochloric acid is 7.84 times of the mass of the arylated cellulose, the paraformaldehyde is 0.38 times of the mass of the arylated cellulose, the temperature is controlled at 20 ℃, phosphorus oxychloride with the mass fraction of 0.45 time of the mass of the arylated cellulose is dropwise added at the speed of 0.65mL/min, reacting for 18 hours at 330rpm, performing suction filtration, washing for 8 times by using deionized water and a sodium bicarbonate aqueous solution in sequence, wherein the mass ratio of sodium bicarbonate to deionized water is 1:32, washing with distilled water until the pH of the solution is 6.5, 60 ℃ and 0.02MPa, and performing vacuum drying for 2.5 hours to obtain cellulose chloride;
c. adding chlorinated cellulose and ethyl acetate with the mass 3.27 times of that of the chlorinated cellulose into a reaction bottle, stirring and dissolving, adding dodecyl dimethylamine with the mass 0.67 time of that of the chlorinated cellulose under the cooling of ice-water bath at the temperature of 1 ℃, reacting for 22 hours at room temperature, and filtering to obtain modified cellulose;
d. placing polyvinylidene fluoride in a vacuum oven with the temperature of 100 ℃ and the pressure of 0.08MPa for drying for 23h, adding triethyl phosphate with the mass of 2.45 times that of the polyvinylidene fluoride, stirring for 4.2h at 240rpm, placing in an outer-layer spinning feed liquid tank, standing and defoaming for 13 h; adding modified cellulose, dimethylformamide, acetone and glycerol into the inner spinning feed liquid tank according to the mass ratio of 1:1.45:0.88: 3; stirring for 3.5h at 260rpm, standing for defoaming for 9h, and co-extruding to obtain a hollow fiber membrane;
e. the hollow fiber membrane is treated for 7min under the ultrasonic irradiation with the power of 340W at the temperature of 150 ℃, and then is kept stand for 2.5h at room temperature, thus obtaining the sterilization membrane.
Furthermore, in the coextrusion process in the step d, the speed of the spinning solution of the outer layer is 0.2mL/min, the speed of the spinning solution of the inner layer is 0.8mL/min, the speed of the core solution is 0.3mL/min, the temperature of a spray head is 75 ℃, the condensation temperature is 15 ℃, and the condensation source is tap water; the core liquid is formed by mixing methyl pyrrolidone and deionized water according to the volume ratio of 95: 5.
Further, the cold soaking temperature of the spray freeze drying in the step (7) is-85 ℃, and the feeding amount is 13 mL/min.
Comparative example 3
A method for extracting elastin by using bovine heart vessels mainly comprises the following preparation steps:
(1) placing the bovine heart vessels and purified water with the mass of 4.2 times of the bovine heart vessels in a colloid mill pulverizer, and pulverizing for 4min to obtain slurry;
(2) placing the slurry in a glass container, adding 5% by mass of sodium hydroxide until the pH of the solution is 7.2, placing the slurry in a microwave oven with the microwave power of 630W, heating for 60s, and cooling to room temperature to obtain a heated extracting solution;
(3) placing the heated extract in an ultrasonic cleaning machine with ultrasonic power of 150W, adding 0.03 times of hydrolytic protease of bovine heart vessel, and extracting for 50min to obtain enzymolysis extract;
(4) placing the enzymolysis extractive solution in a centrifuge, centrifuging at 4350rpm for 13min, placing in 50 deg.C constant temperature water bath, adding neutral protease 0.01 times the mass of heart vessel of cattle, performing enzymolysis for 10min, adding macroporous resin 0.1 times the mass of heart vessel of cattle, adsorbing for 43min, and filtering to obtain supernatant;
(5) heating the supernatant to 88 ℃, inactivating enzyme for 20min, and adding 5% hydrochloric acid by mass until the pH of the solution is 4.9; then placing the mixture in a centrifuge, and centrifuging the mixture for 14min at 6150rpm to obtain elastin;
(6) sterilizing the elastin by a self-made sterilization membrane;
(7) and (4) carrying out spray freeze drying on the sterilized elastin to finish the elastin extraction by utilizing the bovine heart vessels.
Further, the linear speed of a rotor in the colloid mill pulverizer in the step (1) is 25 m/s.
Further, the preparation method of the self-made sterilization membrane in the step (6) comprises the following steps:
a. dissolving cellulose in deionized water with the mass of 43.2 times of that of the cellulose, ultrasonically dissolving the cellulose for 4min at 230W, heating to 90 ℃, adding dodecyl phthalate with the mass of 1.36 times of that of the cellulose, stirring the mixture for 25min at 260rpm, reacting the mixture for 53min at 276W ultrasonic power, cooling to room temperature, and rotationally steaming the mixture for 4h at 300rpm and 80 ℃ to obtain arylated cellulose;
b. adding arylated cellulose into a three-neck flask, stirring at the speed of 50rpm, adding hydrochloric acid with the mass fraction of 65% and paraformaldehyde with the mass fraction of 0.38 times of the mass of the arylated cellulose, wherein the hydrochloric acid is 7.84 times of the mass of the arylated cellulose, the paraformaldehyde is 0.38 times of the mass of the arylated cellulose, the temperature is controlled at 20 ℃, phosphorus oxychloride with the mass fraction of 0.45 time of the mass of the arylated cellulose is dropwise added at the speed of 0.65mL/min, reacting for 18 hours at 330rpm, performing suction filtration, washing for 8 times by using deionized water and a sodium bicarbonate aqueous solution in sequence, wherein the mass ratio of sodium bicarbonate to deionized water is 1:32, washing with distilled water until the pH of the solution is 6.5, 60 ℃ and 0.02MPa, and performing vacuum drying for 2.5 hours to obtain cellulose chloride;
c. adding chlorinated cellulose and ethyl acetate with the mass 3.27 times of that of the chlorinated cellulose into a reaction bottle, stirring and dissolving, adding dodecyl dimethylamine with the mass 0.67 time of that of the chlorinated cellulose under the cooling of ice-water bath at the temperature of 1 ℃, reacting for 22 hours at room temperature, and filtering to obtain modified cellulose;
d. placing polyvinylidene fluoride in a vacuum oven with the temperature of 100 ℃ and the pressure of 0.08MPa for drying for 23h, adding triethyl phosphate with the mass of 2.45 times that of the polyvinylidene fluoride, stirring for 4.2h at 240rpm, placing in an outer-layer spinning feed liquid tank, standing and defoaming for 13 h; adding modified cellulose, dimethylformamide, acetone and glycerol into the inner spinning feed liquid tank according to the mass ratio of 1:1.45:0.88: 3; stirring for 3.5h at 260rpm, standing for defoaming for 9h, and co-extruding to obtain a hollow fiber membrane;
e. the hollow fiber membrane is treated for 7min under the ultrasonic irradiation with the power of 340W at the temperature of 150 ℃, and then is kept stand for 2.5h at room temperature, thus obtaining the sterilization membrane.
Furthermore, in the coextrusion process in the step d, the speed of the spinning solution of the outer layer is 0.2mL/min, the speed of the spinning solution of the inner layer is 0.8mL/min, the speed of the core solution is 0.3mL/min, the temperature of a spray head is 75 ℃, the condensation temperature is 15 ℃, and the condensation source is tap water; the core liquid is formed by mixing methyl pyrrolidone and deionized water according to the volume ratio of 95: 5.
Further, the cold soaking temperature of the spray freeze drying in the step (7) is-85 ℃, and the feeding amount is 13 mL/min.
Comparative example 4
A method for extracting elastin by using bovine heart vessels mainly comprises the following preparation steps:
(1) placing the bovine heart vessels and purified water with the mass of 4.2 times of the bovine heart vessels in a colloid mill pulverizer, and pulverizing for 4min to obtain slurry;
(2) placing the slurry in a glass container, adding 5% by mass of sodium hydroxide until the pH of the solution is 7.2, placing the slurry in a microwave oven with the microwave power of 630W, heating for 60s, and cooling to room temperature to obtain a heated extracting solution;
(3) placing the heated extract in an ultrasonic cleaning machine with ultrasonic power of 150W, adding 0.03 times of hydrolytic protease of bovine heart vessel, and extracting for 50min to obtain enzymolysis extract;
(4) placing the enzymolysis extractive solution in a centrifuge, centrifuging at 4350rpm for 13min, placing in 50 deg.C constant temperature water bath, adding neutral protease 0.01 times the mass of heart vessel of cattle, performing enzymolysis for 10min, adding macroporous resin 0.1 times the mass of heart vessel of cattle, adsorbing for 43min, and filtering to obtain supernatant;
(5) placing the supernatant in an infrared generator with infrared energy intensity of 4kW/m2Inactivating enzyme for 7min, and adding 5% hydrochloric acid until pH is 4.9; then placing the mixture in a centrifuge, and centrifuging the mixture for 14min at 6150rpm to obtain elastin;
(6) sterilizing the elastin by a polyvinylidene fluoride membrane;
(7) and (4) carrying out spray freeze drying on the sterilized elastin to finish the elastin extraction by utilizing the bovine heart vessels.
Further, the linear speed of a rotor in the colloid mill pulverizer in the step (1) is 25 m/s.
Further, the infrared generator in the step (5) is preheated for 20min before being used, and the temperature reaches 180 ℃.
Further, the cold soaking temperature of the spray freeze drying in the step (7) is-85 ℃, and the feeding amount is 13 mL/min.
Comparative example 5
A method for extracting elastin by using bovine heart vessels mainly comprises the following preparation steps:
(1) placing the bovine heart vessels and purified water with the mass of 4.2 times of the bovine heart vessels in a colloid mill pulverizer, and pulverizing for 4min to obtain slurry;
(2) placing the slurry in a glass container, adding 0.03 times of hydrolytic protease of bovine heart vessel, performing enzymolysis for 8h to obtain an enzymolysis extract, heating to 88 deg.C, inactivating enzyme for 20min, and adding 5% hydrochloric acid until the pH of the solution is 4.9 to obtain elastin;
(3) standing elastin for 4h, and taking supernatant; filtering the supernatant with cotton cake filter, adding active carbon 0.0001 times the weight of the supernatant, heating to 80 deg.C, maintaining the temperature for 90min, and filtering with plate-and-frame filter to obtain decolorized protein;
(4) sterilizing the decolorized protein by a polyvinylidene fluoride membrane;
(5) and (4) carrying out spray freeze drying on the sterilized elastin to finish the elastin extraction by utilizing the bovine heart vessels.
Further, the linear speed of a rotor in the colloid mill pulverizer in the step (1) is 25 m/s.
Further, the cold soaking temperature of the spray freeze drying in the step (5) is-85 ℃, and the feeding amount is 13 mL/min.
Examples of effects
The following table 1 shows the results of performance analysis of elastin extraction using bovine heart vessels using examples 1 to 2 of the present invention and comparative examples 1 to 6.
TABLE 1
| Example 1 | Example 2 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 | |
| Extraction ratio (%) | 89.1 | 89.0 | 77.3 | 70.6 | 76.1 | 75.0 | 70.1 |
| Purity (%) | 96.1 | 96.0 | 89.9 | 81.7 | 83.1 | 81.2 | 80.9 |
| Total number of colonies (g) | 5.1 | 5.0 | 7.3 | 7.5 | 7.2 | 90.6 | 92.2 |
| Coliform group (g) | 0.7 | 0.6 | 1.9 | 2.1 | 2.1 | 12.6 | 13.7 |
| Mold (g) | 1.2 | 1.1 | 3.3 | 3.9 | 4.4 | 31.9 | 33.0 |
From the comparison of the experimental data of examples 1 and 2 and comparative example 5, it can be found that the extraction process utilizes ultrasonic wave to assist the enzyme extraction, the time consumption is short, and the extraction rate is high; neutral protease is combined with macroporous resin for decolorization, so that the method is efficient and rapid, and the loss of elastin is reduced; the self-made sterilization membrane is utilized, and the bacteria content in the elastin is reduced through the combined action of physics and chemistry; from the comparison of the experimental data of examples 1 and 2 and comparative example 1, it can be found that in the extraction process, if ultrasonic wave is not used for assistance, the cavitation effect and the mechanical effect of the ultrasonic wave cannot be utilized, the cell wall cannot be broken, the dissolution rate of the elastin is reduced, the elastin cannot be fully contacted with the hydrolytic protease, the elastin is not fully extracted, and the extraction process consumes too much time; from the comparison of experimental data of examples 1 and 2 and comparative example 2, it can be seen that, if cotton cake and activated carbon are used for color removal in the extraction process, the activated carbon is easy to destroy the elastin structure, resulting in elastin loss, and meanwhile, cotton wool and activated carbon in the cotton cake are easy to remain in elastin in the adsorption and decoloration process, reducing the purity of elastin; from the comparison of the experimental data of the examples 1 and 2 and the comparative example 3, it can be found that if infrared enzyme deactivation is not used in the extraction process, and high-temperature enzyme deactivation is adopted, the time consumption is long, meanwhile, high temperature easily causes the deformation of elastin, and the purity of elastin is reduced; from the comparison of the experimental data of examples 1 and 2 and comparative example 4, it can be found that the self-made sterilization membrane is not used in the extraction process, the elastin cannot be efficiently sterilized by the antibacterial group, only part of the bacteria can be removed by physical filtration, the microbe content cannot reach the standard, and the self-made sterilization membrane is not used, so that the high hydrophobicity of the self-made sterilization membrane cannot be utilized to concentrate the elastin, thereby affecting the concentration of the elastin.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (6)
1. A method for extracting elastin by using bovine heart vessels is characterized by mainly comprising the following preparation steps:
(1) placing the bovine heart vessels and purified water with the mass of 3-5 times of the bovine heart vessels into a colloid mill pulverizer, and pulverizing for 3-5 min to obtain slurry;
(2) placing the slurry in a glass container, adding 5% by mass of sodium hydroxide until the pH of the solution is 7.0-7.5, placing the solution in a microwave oven with the microwave power of 600-700W, heating for 50-70 s, and cooling to room temperature to obtain a heating extracting solution;
(3) putting the heated extracting solution into an ultrasonic cleaning machine, adding hydrolysis protease with the ultrasonic power of 140-160W, wherein the mass of the hydrolysis protease is 0.03 times that of bovine heart vessels, and extracting for 40-60 min to obtain an enzymolysis extracting solution;
(4) placing the enzymolysis extracting solution into a centrifuge, centrifuging at 4000-4500 rpm for 10-15 min, placing into a 50 ℃ constant temperature water bath kettle, adding neutral protease with the mass of 0.01 time of that of bovine heart vessels, performing enzymolysis for 9-11 min, adding macroporous resin with the mass of 0.1 time of that of the bovine heart vessels, adsorbing for 40-50 min, and filtering to obtain a supernatant;
(5) placing the supernatant in an infrared generator, wherein the infrared energy intensity is 3-5 kW/m2Inactivating enzyme for 6-8 min, and adding 5% by mass of hydrochloric acid until the pH of the solution is 4.8-5.0; then placing the mixture in a centrifuge, and centrifuging the mixture at 6000-6200 rpm for 13-15 min to obtain elastin;
(6) sterilizing the elastin by a self-made sterilization membrane;
(7) and (4) carrying out spray freeze drying on the sterilized elastin to finish the elastin extraction by utilizing the bovine heart vessels.
2. The method for extracting elastin by using bovine heart vessel as claimed in claim 1, wherein the linear speed of rotor in the colloid mill pulverizer of step (1) is 20-30 m/s.
3. The method for extracting elastin by using bovine heart vessel as claimed in claim 2, wherein the infrared generator in step (5) is preheated for 20min before use, and the temperature reaches 180 ℃.
4. The method for extracting elastin by using bovine heart vessel as claimed in claim 3, wherein the preparation method of the self-made degerming membrane in step (6) is as follows:
a. dissolving cellulose in deionized water with the mass 40-45 times of that of the cellulose, ultrasonically dissolving for 3-5 min at 200-250W, heating to 80-100 ℃, adding dodecyl phthalate with the mass 1.2-1.5 times of that of the cellulose, stirring for 20-30 min at 200-300 rpm, reacting for 50-60 min at 200-300W ultrasonic power, cooling to room temperature, and rotationally steaming for 3-5 h at 300rpm and 80 ℃ to obtain arylated cellulose;
b. adding arylated cellulose into a three-necked flask, stirring at 50rpm, adding hydrochloric acid with the mass fraction of 65% and paraformaldehyde with the mass fraction of 0.35-0.4 time of the arylated cellulose, wherein the hydrochloric acid is 7.5-8 times of the mass of the arylated cellulose, the paraformaldehyde is 0.35-0.4 time of the mass of the arylated cellulose, the temperature is controlled at 15-25 ℃, phosphorus oxychloride with the mass fraction of 0.4-0.5 time of the arylated cellulose is dropwise added at the speed of 0.5-0.7 mL/min, reacting for 16-20 hours at 300-400 rpm, performing suction filtration, washing with deionized water and a sodium bicarbonate aqueous solution for 7-10 times in sequence, wherein the mass ratio of sodium bicarbonate to deionized water in the sodium bicarbonate aqueous solution is 1:32, washing with distilled water until the pH of the solution is 6-7, 60 ℃ and 0.02MPa, and performing vacuum drying for 2-3 hours to obtain chlorinated cellulose;
c. adding chlorinated cellulose and ethyl acetate which is 3-3.5 times of the mass of the chlorinated cellulose into a reaction bottle, stirring for dissolving, adding dodecyl dimethylamine which is 0.6-0.7 times of the mass of the chlorinated cellulose under cooling in ice-water bath at 0-2 ℃, reacting for 20-24 h at room temperature, and filtering to obtain modified cellulose;
d. placing polyvinylidene fluoride in a vacuum oven with the temperature of 100 ℃ and the pressure of 0.08MPa for drying for 20-24 h, adding triethyl phosphate with the mass being 2-3 times that of the polyvinylidene fluoride, stirring for 4-5 h at 200-300 rpm, placing in an outer spinning feed liquid tank, and standing and defoaming for 12-14 h; adding modified cellulose, dimethylformamide, acetone and glycerol into the inner spinning feed liquid tank according to the mass ratio of 1:1.45:0.88: 3; stirring at 200-300 rpm for 2-4 h, standing for defoaming for 8-10 h, and co-extruding to obtain a hollow fiber membrane;
e. and (3) treating the hollow fiber membrane for 5-10 min under ultrasonic irradiation with power of 300-400W at 145-155 ℃, standing for 2-3 h at room temperature, and thus obtaining the antibacterial membrane.
5. The method for extracting elastin by using bovine heart vessel as claimed in claim 4, wherein during the co-extrusion process of step d, the outer layer spinning solution speed is 0.2mL/min, the inner layer spinning solution speed is 0.8mL/min, and the core solution speed is 0.3 mL/min; the temperature of the spray head is 75 ℃, the condensation temperature is 15 ℃, and the condensation source is tap water; the core liquid is formed by mixing methyl pyrrolidone and deionized water according to the volume ratio of 95: 5.
6. The method for extracting elastin by using bovine heart vessel as claimed in claim 5, wherein the cold dipping temperature of the spray freeze drying in the step (7) is-90 to-80 ℃, and the feeding amount is 10 to 15 mL/min.
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| Publication number | Priority date | Publication date | Assignee | Title |
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