CN111499620A - Method for extracting puerarin in wastewater by using puerarin aptamer - Google Patents
Method for extracting puerarin in wastewater by using puerarin aptamer Download PDFInfo
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- B01D15/3819—Affinity chromatography of the nucleic acid-nucleic acid binding protein type
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Abstract
The invention provides a method for extracting puerarin in wastewater by using puerarin aptamer, which comprises the following steps: the method comprises the following steps of wastewater pretreatment, primary concentration, ultrasonic wave removal, nucleic acid aptamer preparation, high-affinity target molecule combination nucleic acid aptamer, primary purification, secondary purification and finished product preparation. The invention has the advantages that: the method for extracting puerarin in wastewater by using the puerarin aptamer provided by the invention has the advantages that the puerarin aptamer with high specificity is assisted on the basis of conventional mechanical purification, the characteristic that the aptamer has high affinity to puerarin is utilized, the polymerization adsorption of the aptamer to a puerarin target and the enrichment of puerarin are realized, the separation of puerarin from wastewater is accelerated, the extraction speed and efficiency are effectively improved, and the high-efficiency extraction of puerarin in wastewater is finally realized.
Description
Technical Field
The invention relates to the technical field of separation and extraction, in particular to a method for extracting puerarin in wastewater by using puerarin aptamer.
Background
Puerarin is also known as puerarin. Is isoflavone derivative separated from radix Puerariae with dilating coronary effect. Is present in root of Pueraria lobata Ohwi of Leguminosae. Has antipyretic, tranquilizing and coronary blood flow increasing effects, and can be used for treating coronary heart disease, angina pectoris, and hypertension.
Puerarin has good curative effect on cardiovascular and cerebrovascular diseases such as coronary heart disease, angina pectoris and the like, and the purified product is very common radix puerariae, so that the purification methods widely accepted at present at home comprise a water bath reflux method, an ultrasonic purification method and a microwave extraction method, however, the existing puerarin extraction method can be smashed and filtered in the extraction process regardless of which method is adopted, the generated filtrate also contains a lot of puerarin which is not extracted, and the direct abandonment of the puerarin causes the waste of raw materials, which is also the problem of lower purity and utilization rate in the puerarin extraction process at present, so the invention develops a brand-new method for extracting puerarin in waste water by using a puerarin nucleic acid aptamer aiming at the problems.
Disclosure of Invention
In order to make up the defects of the prior art, the invention provides a brand-new method for extracting puerarin in wastewater by using a puerarin aptamer.
The invention provides a method for extracting puerarin in wastewater by using puerarin aptamer, which comprises the following steps:
(1) wastewater pretreatment: filtering impurities in the wastewater for producing the pueraria starch, specifically, adding an organic flocculant with the mass fraction of 1-3% into the wastewater, standing for 20-30min, removing floating materials, and then sieving by a sieve to remove impurities to obtain a pretreatment filtrate for later use;
(2) primary concentration: distilling the pretreated filtrate, specifically heating the filtrate to 60-70 ℃ in a low-pressure state by using a distillation container, and carrying out heat preservation and heating for 30-40min to evaporate water in the pretreated filtrate to obtain residue I;
(3) ultrasonic breaking: putting the first residue into ultrasonic wave breaking equipment, and starting the ultrasonic wave breaking equipment to generate ultrasonic waves to break cell walls to obtain a wall breaking product, wherein the ultrasonic wave breaking time is 10-20 min; adding the wall-broken product into an ethanol water solution with the mass coefficient concentration of 60-80%, heating to 40-55 ℃, stirring for 1-2h, fully leaching, and filtering to remove insoluble impurities to obtain leaching filtrate; then heating the leaching filtrate to 60 ℃ under a low-pressure state, and evaporating to dryness to obtain residue II;
(4) preparation of aptamer: constructing a large-capacity aptamer library under the vacuum sterile condition; then, puerarin is used as a target to interact with the aptamer library to obtain a binding molecule; separating the aptamer in the binding molecule from the target by adopting a nucleotide separation method, and enriching the aptamer by utilizing a photoconductive relay reaction;
(5) high affinity target molecules bind nucleic acid aptamers: repeating the operation of the step (4) for multiple times to obtain a target molecule binding nucleic acid aptamer with high affinity for the puerarin target, and preparing the target molecule binding nucleic acid aptamer solution with high affinity with the mass fraction of 1-5% by adopting a TE buffer solution;
(6) primary purification: adding a solid phase carrier into the solution of the target molecule binding aptamer with high affinity, and fixing the target molecule binding aptamer with high affinity onto the solid phase carrier through a coupling reaction to obtain a solid phase carrier binding body; adding a dissolving solution with the mass 5 times that of the residue II prepared in the step (3) into the residue II to fully dissolve the residue to obtain a solution; then reacting the solution with the solid phase carrier combination, stirring for 20-30min at 30-50 ℃ to complete the reaction, and separating out buffer solution in the reaction system to obtain the solid phase carrier combination for adsorbing puerarin; washing the solid phase carrier combination for 3 times, adding the washed solid phase carrier into hot water of 90-100 deg.C, and shaking for 5min to obtain a first puerarin water solution and a first analytic puerarin solid phase carrier;
(7) and (3) secondary purification: reacting the buffer solution in the step (6) with the analytic puerarin solid phase carrier again, stirring for 30-50min at 30-50 ℃ to complete reaction, separating out secondary buffer solution in a reaction system to obtain a secondary puerarin solid phase carrier combination, washing the secondary puerarin solid phase carrier combination for 3 times, adding the washed secondary puerarin solid phase carrier into hot water at 90-100 ℃, and shaking for 5min to obtain a secondary puerarin water solution and a secondary analytic puerarin solid phase carrier; mixing the puerarin water solution I and the puerarin water solution II to obtain a puerarin water solution;
(8) and (3) preparing a finished product: and (4) putting the puerarin water solution prepared in the step (7) into a vacuum drying oven, and drying the puerarin water solution at the temperature of between 60 and 70 ℃ in vacuum to constant weight to obtain a puerarin finished product.
As a preferred scheme, the screen in the step (1) is a 50-mesh screen.
Preferably, the nucleotide sequence of the target molecule with high affinity for the puerarin target prepared in step (5) binding aptamer is shown as SEQ ID No. 1.
Preferably, the nucleotide sequence of the target molecule with high affinity for the puerarin target prepared in step (5) binding aptamer is shown as SEQ ID No. 2.
Preferably, the ultrasonic wave breaking time in the step (3) is 16min, and the ultrasonic wave breaking temperature is 67 ℃; the temperature of the stirring extraction is 50 ℃, and the stirring time is 1.4 h.
Preferably, in the step (2), the filtrate is heated to 65 ℃ under a low pressure state by using a distillation container, and the water in the pretreated filtrate is evaporated by heat preservation and heating for 35min to obtain residue I.
Preferably, the multiple repetitions of step (5) are 7 repetitions, and the mass fraction of the target molecule with high affinity bound to the aptamer solution is 3%.
Preferably, the composition of the dissolution solution in the step (6) is 20 mmol/L Tris-HCl, 100 mmol/L NaCl, 2 mmol/L MgCl25 mmol/L KCl, 0.02% Tween 20 by mass fraction, 1mg/m L yeast tRNA, pH 7.6, reaction temperature of the solution and the solid phase carrier combination is 32 ℃, and stirring time is 30 min.
Preferably, the reaction temperature of the buffer solution and the solid phase carrier for resolving puerarin again in step (7) is 37 ℃, and the stirring time is 50 min.
Preferably, the vacuum drying temperature in step (8) is 68 ℃.
The invention has the advantages that: the method for extracting puerarin in wastewater by using the puerarin aptamer provided by the invention has the advantages that the puerarin aptamer with high specificity is assisted on the basis of conventional mechanical purification, the characteristic that the aptamer has high affinity to puerarin is utilized, the polymerization adsorption of the aptamer to a puerarin target and the enrichment of puerarin are realized, the separation of puerarin from wastewater is accelerated, the extraction speed and efficiency are effectively improved, and the high-efficiency extraction of puerarin in wastewater is finally realized.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but 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.
Example 1
A method for extracting puerarin in wastewater by using puerarin aptamer comprises the following steps:
(1) wastewater pretreatment: filtering impurities in the wastewater for producing the pueraria starch, specifically, adding an organic flocculant with the mass fraction of 1-3% into the wastewater, standing for 20-30min, removing floating materials, and filtering and removing impurities through a 50-mesh screen to obtain a pretreated filtrate for later use;
(2) primary concentration: distilling the pretreatment filtrate, specifically heating the filtrate to 65 ℃ in a low-pressure state by using a distillation container, and carrying out heat preservation and heating for 35min to evaporate water in the pretreatment filtrate to obtain residue I;
(3) ultrasonic breaking: putting the first residue into ultrasonic wave breaking equipment, and starting the ultrasonic wave breaking equipment to generate ultrasonic waves to break cell walls to obtain a wall-broken product, wherein the ultrasonic wave breaking time is 16min, and the temperature is 67 ℃; adding the wall-broken product into an ethanol water solution with the mass coefficient concentration of 60-70%, heating to 50 ℃, stirring for 1.4h, fully leaching, and filtering to remove insoluble impurities to obtain leaching filtrate; then heating the leaching filtrate to 60 ℃ under a low-pressure state, and evaporating to dryness to obtain residue II;
(4) preparation of aptamer: constructing a large-capacity aptamer library under the vacuum sterile condition; then, puerarin is used as a target to interact with the aptamer library to obtain a binding molecule; separating the aptamer in the binding molecule from the target by adopting a nucleotide separation method, and enriching the aptamer by utilizing a photoconductive relay reaction;
(5) high affinity target molecules bind nucleic acid aptamers: repeating the operation of the step (4) for 7 times to obtain a target molecule binding aptamer with high affinity for the puerarin target, and preparing a target molecule binding aptamer solution with high affinity with a mass fraction of 1% by using a TE buffer solution; and the nucleotide sequence of the target molecule with high affinity for the puerarin target and the aptamer is shown as SEQ ID NO.1 or SEQ ID NO. 2;
(6) repeating the operation of the step (4) for multiple times to obtain the target molecule-bound aptamer with high affinity aiming at the puerarin target, purifying for one time, namely adding a solid phase carrier into the solution of the target molecule-bound aptamer with high affinity, fixing the target molecule-bound aptamer with high affinity onto the solid phase carrier through a coupling reaction to obtain a solid phase carrier combination, and adding a dissolving solution with the mass 5 times that of the residue II prepared in the step (3) (the composition of the dissolving solution is 20 mmol/L-Tris-HCl, 100 mmol/L NaCl and 2 mmol/L MgCl)2The method comprises the following steps of (1) fully dissolving 5 mmol/L KCl, Tween 20 with the mass fraction of 0.02%, 1mg/m L yeast tRNA with the pH value of 7.6) to obtain a solution, reacting the solution with the solid phase carrier combination, stirring for 30min at the temperature of 32 ℃ to complete reaction, separating buffer solution in a reaction system to obtain an adsorption puerarin solid phase carrier combination, washing the adsorption puerarin solid phase carrier combination for 3 times, adding the washed adsorption puerarin solid phase carrier into hot water with the temperature of 90-100 ℃, and shaking for 5min to obtain a puerarin water solution I and an analysis puerarin solid phase carrier I;
(7) and (3) secondary purification: reacting the buffer solution in the step (6) with the analytic puerarin solid phase carrier again, stirring for 50min at 37 ℃ to complete reaction, separating out secondary buffer solution in a reaction system to obtain a secondary puerarin solid phase carrier combination, washing the secondary puerarin solid phase carrier combination for 3 times, adding the washed secondary puerarin solid phase carrier into hot water at 90-100 ℃, and shaking for 5min to obtain a secondary puerarin aqueous solution and a secondary analytic puerarin solid phase carrier; mixing the puerarin water solution I and the puerarin water solution II to obtain a puerarin water solution;
(8) and (3) preparing a finished product: and (4) putting the puerarin water solution prepared in the step (7) into a vacuum drying oven, and drying the puerarin water solution at 68 ℃ in vacuum to constant weight to obtain a puerarin finished product.
Example 2
A method for extracting puerarin in wastewater by using puerarin aptamer comprises the following steps:
(1) wastewater pretreatment: filtering impurities in the wastewater for producing the pueraria starch, specifically, adding an organic flocculant with the mass fraction of 1-3% into the wastewater, standing for 20-30min, removing floating materials, and filtering and removing impurities through a 50-mesh screen to obtain a pretreated filtrate for later use;
(2) primary concentration: distilling the pretreatment filtrate, specifically heating the filtrate to 65 ℃ in a low-pressure state by using a distillation container, and carrying out heat preservation and heating for 35min to evaporate water in the pretreatment filtrate to obtain residue I;
(3) ultrasonic breaking: putting the first residue into ultrasonic wave breaking equipment, and starting the ultrasonic wave breaking equipment to generate ultrasonic waves to break cell walls to obtain a wall-broken product, wherein the ultrasonic wave breaking time is 16min, and the temperature is 67 ℃; adding the wall-broken product into an ethanol water solution with the mass coefficient concentration of 60-70%, heating to 50 ℃, stirring for 1.4h, fully leaching, and filtering to remove insoluble impurities to obtain leaching filtrate; then heating the leaching filtrate to 60 ℃ under a low-pressure state, and evaporating to dryness to obtain residue II;
(4) preparation of aptamer: constructing a large-capacity aptamer library under the vacuum sterile condition; then, puerarin is used as a target to interact with the aptamer library to obtain a binding molecule; separating the aptamer in the binding molecule from the target by adopting a nucleotide separation method, and enriching the aptamer by utilizing a photoconductive relay reaction;
(5) high affinity target molecules bind nucleic acid aptamers: repeating the operation of the step (4) for 7 times to obtain a target molecule binding aptamer with high affinity for the puerarin target, and preparing a target molecule binding aptamer solution with high affinity with a mass fraction of 3% by adopting a TE buffer solution; and the nucleotide sequence of the target molecule with high affinity for the puerarin target and the aptamer is shown as SEQ ID NO.1 or SEQ ID NO. 2;
(6) repeating the operation of the step (4) for multiple times to obtain the target molecule-bound aptamer with high affinity aiming at the puerarin target, purifying for one time, namely adding a solid phase carrier into the solution of the target molecule-bound aptamer with high affinity, fixing the target molecule-bound aptamer with high affinity onto the solid phase carrier through a coupling reaction to obtain a solid phase carrier combination, and adding a dissolving solution with the mass 5 times that of the residue II prepared in the step (3) (the composition of the dissolving solution is 20 mmol/L-Tris-HCl, 100 mmol/L NaCl and 2 mmol/L MgCl)2The method comprises the following steps of (1) fully dissolving 5 mmol/L KCl, Tween 20 with the mass fraction of 0.02%, 1mg/m L yeast tRNA with the pH value of 7.6) to obtain a solution, reacting the solution with the solid phase carrier combination, stirring for 30min at the temperature of 32 ℃ to complete reaction, separating buffer solution in a reaction system to obtain an adsorption puerarin solid phase carrier combination, washing the adsorption puerarin solid phase carrier combination for 3 times, adding the washed adsorption puerarin solid phase carrier into hot water with the temperature of 90-100 ℃, and shaking for 5min to obtain a puerarin water solution I and an analysis puerarin solid phase carrier I;
(7) and (3) secondary purification: reacting the buffer solution in the step (6) with the analytic puerarin solid phase carrier again, stirring for 50min at 37 ℃ to complete reaction, separating out secondary buffer solution in a reaction system to obtain a secondary puerarin solid phase carrier combination, washing the secondary puerarin solid phase carrier combination for 3 times, adding the washed secondary puerarin solid phase carrier into hot water at 90-100 ℃, and shaking for 5min to obtain a secondary puerarin aqueous solution and a secondary analytic puerarin solid phase carrier; mixing the puerarin water solution I and the puerarin water solution II to obtain a puerarin water solution;
(8) and (3) preparing a finished product: and (4) putting the puerarin water solution prepared in the step (7) into a vacuum drying oven, and drying the puerarin water solution at 68 ℃ in vacuum to constant weight to obtain a puerarin finished product.
Example 3
A method for extracting puerarin in wastewater by using puerarin aptamer comprises the following steps:
(1) wastewater pretreatment: filtering impurities in the wastewater for producing the pueraria starch, specifically, adding an organic flocculant with the mass fraction of 1-3% into the wastewater, standing for 20-30min, removing floating materials, and filtering and removing impurities through a 50-mesh screen to obtain a pretreated filtrate for later use;
(2) primary concentration: distilling the pretreatment filtrate, specifically heating the filtrate to 65 ℃ in a low-pressure state by using a distillation container, and carrying out heat preservation and heating for 35min to evaporate water in the pretreatment filtrate to obtain residue I;
(3) ultrasonic breaking: putting the first residue into ultrasonic wave breaking equipment, and starting the ultrasonic wave breaking equipment to generate ultrasonic waves to break cell walls to obtain a wall-broken product, wherein the ultrasonic wave breaking time is 16min, and the temperature is 67 ℃; adding the wall-broken product into an ethanol water solution with the mass coefficient concentration of 60-70%, heating to 50 ℃, stirring for 1.4h, fully leaching, and filtering to remove insoluble impurities to obtain leaching filtrate; then heating the leaching filtrate to 60 ℃ under a low-pressure state, and evaporating to dryness to obtain residue II;
(4) preparation of aptamer: constructing a large-capacity aptamer library under the vacuum sterile condition; then, puerarin is used as a target to interact with the aptamer library to obtain a binding molecule; separating the aptamer in the binding molecule from the target by adopting a nucleotide separation method, and enriching the aptamer by utilizing a photoconductive relay reaction;
(5) high affinity target molecules bind nucleic acid aptamers: repeating the operation of the step (4) for 7 times to obtain a target molecule binding aptamer with high affinity for the puerarin target, and preparing a target molecule binding aptamer solution with high affinity with a mass fraction of 5% by using a TE buffer solution; and the nucleotide sequence of the target molecule with high affinity for the puerarin target and the aptamer is shown as SEQ ID NO.1 or SEQ ID NO. 2;
(6) repeating the operation of the step (4) for multiple times to obtain the target molecule-bound aptamer with high affinity aiming at the puerarin target, purifying for one time, namely adding a solid phase carrier into the solution of the target molecule-bound aptamer with high affinity, fixing the target molecule-bound aptamer with high affinity onto the solid phase carrier through a coupling reaction to obtain a solid phase carrier combination, and adding a dissolving solution with the mass 5 times that of the residue II prepared in the step (3) (the composition of the dissolving solution is 20 mmol/L-Tris-HCl, 100 mmol/L NaCl and 2 mmol/L MgCl)2The method comprises the following steps of (1) fully dissolving 5 mmol/L KCl, Tween 20 with the mass fraction of 0.02%, 1mg/m L yeast tRNA with the pH value of 7.6) to obtain a solution, reacting the solution with the solid phase carrier combination, stirring for 30min at the temperature of 32 ℃ to complete reaction, separating buffer solution in a reaction system to obtain an adsorption puerarin solid phase carrier combination, washing the adsorption puerarin solid phase carrier combination for 3 times, adding the washed adsorption puerarin solid phase carrier into hot water with the temperature of 90-100 ℃, and shaking for 5min to obtain a puerarin water solution I and an analysis puerarin solid phase carrier I;
(7) and (3) secondary purification: reacting the buffer solution in the step (6) with the analytic puerarin solid phase carrier again, stirring for 50min at 37 ℃ to complete reaction, separating out secondary buffer solution in a reaction system to obtain a secondary puerarin solid phase carrier combination, washing the secondary puerarin solid phase carrier combination for 3 times, adding the washed secondary puerarin solid phase carrier into hot water at 90-100 ℃, and shaking for 5min to obtain a secondary puerarin aqueous solution and a secondary analytic puerarin solid phase carrier; mixing the puerarin water solution I and the puerarin water solution II to obtain a puerarin water solution;
(8) and (3) preparing a finished product: and (4) putting the puerarin water solution prepared in the step (7) into a vacuum drying oven, and drying the puerarin water solution at 68 ℃ in vacuum to constant weight to obtain a puerarin finished product.
Comparative example 1
A method for extracting puerarin in wastewater by using puerarin aptamer comprises the following steps:
(1) wastewater pretreatment: filtering impurities in the wastewater for producing the pueraria starch, specifically, adding an organic flocculant with the mass fraction of 1-3% into the wastewater, standing for 20-30min, removing floating materials, and filtering and removing impurities through a 50-mesh screen to obtain a pretreated filtrate for later use;
(2) primary concentration: distilling the pretreatment filtrate, specifically heating the filtrate to 65 ℃ in a low-pressure state by using a distillation container, and carrying out heat preservation and heating for 35min to evaporate water in the pretreatment filtrate to obtain residues;
(3) leaching: adding the residues into an ethanol water solution with the mass coefficient concentration of 60-70%, heating to 50 ℃, stirring for 1.4h, fully leaching, and filtering to remove insoluble impurities to obtain leaching filtrate; and then heating the leaching filtrate to 60 ℃ under a low pressure state, and evaporating to dryness to obtain a puerarin finished product.
Example 4
Each of the puerarin finished products prepared in example 1, example 2, example 3 and comparative example 1 was added with 10 parts of water, stirred to dissolve, and the concentration of the puerarin was measured, and the results are shown in the following table.
It can be known by synthesizing multiple comparisons, the using amount of the puerarin aptamer has the best cost effectiveness ratio when the using amount is 3% of the mass fraction, puerarin purification is carried out according to the method, in the waste liquid of puerarin production, the puerarin aptamer with high specificity is assisted on the basis of conventional purification, the characteristic that the aptamer has high affinity to puerarin is utilized, the puerarin target can be polymerized and adsorbed by the aptamer, the puerarin enrichment accelerates the precipitation of puerarin in the solution, the purification speed and efficiency can be effectively improved, puerarin which is not easy to be polymerized in the solution can be polymerized, thereby extracting the residual puerarin in the waste liquid, and the utilization rate of raw materials is improved.
It should be understood that the above-described specific embodiments are merely illustrative of the present invention and are not intended to limit the present invention. Obvious variations or modifications which are within the spirit of the invention are possible within the scope of the invention.
Sequence listing
<110> Liaoning science and technology institute
<120> a method for extracting puerarin in wastewater by using puerarin aptamer
<130>2020.04.11
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<170>SIPOSequenceListing 1.0
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<213> Artificial sequence (Artificial sequence)
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cgacgtacgt gcgtccgtac tctggtctgt cgg 33
Claims (10)
1. A method for extracting puerarin in wastewater by using puerarin aptamer is characterized by comprising the following steps:
(1) wastewater pretreatment: filtering impurities in the wastewater for producing the pueraria starch, specifically, adding an organic flocculant with the mass fraction of 1-3% into the wastewater, standing for 20-30min, removing floating materials, and then sieving by a sieve to remove impurities to obtain a pretreatment filtrate for later use;
(2) primary concentration: distilling the pretreated filtrate, specifically heating the filtrate to 60-70 ℃ in a low-pressure state by using a distillation container, and carrying out heat preservation and heating for 30-40min to evaporate water in the pretreated filtrate to obtain residue I;
(3) ultrasonic breaking: putting the first residue into ultrasonic wave breaking equipment, and starting the ultrasonic wave breaking equipment to generate ultrasonic waves to break cell walls to obtain a wall breaking product, wherein the ultrasonic wave breaking time is 10-20 min; adding the wall-broken product into an ethanol water solution with the mass coefficient concentration of 60-80%, heating to 40-55 ℃, stirring for 1-2h, fully leaching, and filtering to remove insoluble impurities to obtain leaching filtrate; then heating the leaching filtrate to 60 ℃ under a low-pressure state, and evaporating to dryness to obtain residue II;
(4) preparation of aptamer: constructing a large-capacity aptamer library under the vacuum sterile condition; then, puerarin is used as a target to interact with the aptamer library to obtain a binding molecule; separating the aptamer in the binding molecule from the target by adopting a nucleotide separation method, and enriching the aptamer by utilizing a photoconductive relay reaction;
(5) high affinity target molecules bind nucleic acid aptamers: repeating the operation of the step (4) for multiple times to obtain a target molecule binding nucleic acid aptamer with high affinity for the puerarin target, and preparing the target molecule binding nucleic acid aptamer solution with high affinity with the mass fraction of 1-5% by adopting a TE buffer solution;
(6) primary purification: adding a solid phase carrier into the solution of the target molecule binding aptamer with high affinity, and fixing the target molecule binding aptamer with high affinity onto the solid phase carrier through a coupling reaction to obtain a solid phase carrier binding body; adding a dissolving solution with the mass 5 times that of the residue II prepared in the step (3) into the residue II to fully dissolve the residue to obtain a solution; then reacting the solution with the solid phase carrier combination, stirring for 20-30min at 30-50 ℃ to complete the reaction, and separating out buffer solution in the reaction system to obtain the solid phase carrier combination for adsorbing puerarin; washing the solid phase carrier combination for 3 times, adding the washed solid phase carrier into hot water of 90-100 deg.C, and shaking for 5min to obtain a first puerarin water solution and a first analytic puerarin solid phase carrier;
(7) and (3) secondary purification: reacting the buffer solution in the step (6) with the analytic puerarin solid phase carrier again, stirring for 30-50min at 30-50 ℃ to complete reaction, separating out secondary buffer solution in a reaction system to obtain a secondary puerarin solid phase carrier combination, washing the secondary puerarin solid phase carrier combination for 3 times, adding the washed secondary puerarin solid phase carrier into hot water at 90-100 ℃, and shaking for 5min to obtain a secondary puerarin water solution and a secondary analytic puerarin solid phase carrier; mixing the puerarin water solution I and the puerarin water solution II to obtain a puerarin water solution;
(8) and (3) preparing a finished product: and (4) putting the puerarin water solution prepared in the step (7) into a vacuum drying oven, and drying the puerarin water solution at the temperature of between 60 and 70 ℃ in vacuum to constant weight to obtain a puerarin finished product.
2. The method for extracting puerarin in wastewater by using puerarin aptamer according to claim 1, wherein the screen in step (1) is a 50-mesh screen.
3. The method for extracting puerarin in wastewater by using puerarin aptamer according to claim 2, wherein the nucleotide sequence of the high-affinity target molecule binding aptamer for puerarin target prepared in step (5) is shown in SEQ ID No. 1.
4. The method for extracting puerarin in wastewater by using puerarin aptamer according to claim 2, wherein the nucleotide sequence of the target molecule with high affinity for puerarin target prepared in step (5) and the aptamer is shown in SEQ ID No. 2.
5. The method for extracting puerarin in wastewater with puerarin aptamer according to claim 3 or 4, wherein the ultrasonic disruption time in step (3) is 16min, and the ultrasonic disruption temperature is 67 ℃; the temperature of the stirring extraction is 50 ℃, and the stirring time is 1.4 h.
6. The method for extracting puerarin in wastewater by using puerarin aptamer according to claim 5, wherein the filtrate obtained in step (2) is heated to 65 ℃ under low pressure by using a distillation container, and the water in the pretreated filtrate is evaporated by heat preservation for 35min to obtain residue I.
7. The method for extracting puerarin in wastewater with puerarin aptamer according to claim 6, wherein the multiple repetitions in step (5) are 7 repetitions; the mass fraction of the high affinity target molecule bound to the aptamer solution was 3%.
8. The method of claim 7, wherein the composition of the solution in step (6) is 20 mmol/L Tris-HCl, 100 mmol/L NaCl, 2 mmol/L MgCl25 mmol/L KCl, 0.02% Tween 20 by mass fraction, 1mg/m L yeast tRNA, pH 7.6, reaction temperature of the solution and the solid phase carrier combination is 32 ℃, and stirring time is 30 min.
9. The method for extracting puerarin from wastewater with puerarin aptamer according to claim 8, wherein the reaction temperature of the buffer solution and the solid phase carrier for re-resolving puerarin in step (7) is 37 ℃ and the stirring time is 50 min.
10. The method for extracting puerarin from wastewater with puerarin aptamer according to claim 9, wherein the vacuum drying temperature in step (8) is 68 ℃.
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