CN117384171A - Method for preparing heme by enzymolysis and alcohol extraction - Google Patents
Method for preparing heme by enzymolysis and alcohol extraction Download PDFInfo
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- GPRXGEKBQVXWAQ-UHFFFAOYSA-L disodium;3-[18-(2-carboxylatoethyl)-8,13-bis(ethenyl)-3,7,12,17-tetramethyl-22,23-dihydroporphyrin-2-yl]propanoate Chemical compound [Na+].[Na+].N1C(C=C2C(=C(C)C(=CC=3C(C)=C(CCC([O-])=O)C(N=3)=C3)N2)C=C)=C(C)C(C=C)=C1C=C1C(C)=C(CCC([O-])=O)C3=N1 GPRXGEKBQVXWAQ-UHFFFAOYSA-L 0.000 description 1
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- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
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- VRIVJOXICYMTAG-IYEMJOQQSA-L iron(ii) gluconate Chemical compound [Fe+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O VRIVJOXICYMTAG-IYEMJOQQSA-L 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of heme preparation, and discloses a method for preparing heme by enzymolysis alcohol extraction, which comprises the following preparation steps: s1: mixing pig blood globulin powder with water, regulating pH value to be alkaline, adding compound proteolytic enzyme for enzymolysis, regulating pH value of enzymolysis liquid to be weak acid, and collecting precipitate; heating water for stirring and washing the precipitate, and centrifugally collecting the precipitate; adding a proper amount of water for suspension, and spray-drying to obtain a heme extraction crude product; s2: adding the heme extraction crude product into alkaline ethanol solution, stirring and extracting, regulating pH by using centrifugate to precipitate heme, and centrifugally collecting heme purification precipitate; s3: the purified precipitate of heme is crystallized by glacial acetic acid, washed by water, washed by alcohol, filtered and spray dried to obtain pure heme crystal. The method enriches heme by recovering protein peptide through enzymolysis, and prepares heme through purification and crystallization, so that the hemoglobin can be comprehensively utilized, the production cost is greatly reduced, the heme with high purity is prepared, and the environmental pollution of the traditional method is avoided.
Description
Technical Field
The invention relates to the technical field of heme extraction, in particular to a method for preparing heme by enzymolysis and alcohol extraction.
Background
Heme (heme), also known as iron porphyrin, is complexed with one iron ion by protoporphyrin IX, and is widely found in animal blood, muscle and certain plant tissues, and is a prosthetic group of hemoglobin, myoglobin, cytochrome C, catalase, peroxidase, etc. Heme is present in blood mainly in erythrocytes, and one molecule of globin is non-covalently bound to four molecules of heme. Heme has important physiological functions and high application value, and can be widely applied to the industries of medicines, foods, chemical engineering and health care products. Clinically, heme is an iron supplement with good curative effect for treating iron deficiency anemia at present, and has the advantages of high bioavailability, no iron accumulation poisoning in vivo, no gastrointestinal irritation and the like compared with common chemical iron supplements such as ferrous sulfate, ferrous fumarate, ferrous gluconate and the like; heme in combination with EDTA can also be used to treat lead poisoning. In the pharmaceutical industry, heme is a raw material for preparing hematoporphyrin drugs for treating malignant tumors and protoporphyrin sodium for treating acute and chronic persistent and chronic active hepatitis. In the food industry, heme is a natural pigment, can replace nitrite serving as a color former and an artificially synthesized pigment which endanger human health in cooked meat products, reduces the carcinogenesis of nitrite, and can keep the natural color inherent to muscles. Heme as a high-efficiency iron source substance has been used in feed production and becomes a novel green feed additive.
The pig blood is about 200 ten thousand tons per year, and is mainly and simply processed into plasma powder and blood cell powder which are used as animal feed, but the pig blood cell powder is rich in protein and heme, has heavy fishy smell and deep color and is difficult to digest and absorb, and the high-quality protein and heme of the pig blood cell powder are not fully utilized and developed, so that the resource waste and the environmental pollution are caused. The pig blood resource is comprehensively developed and utilized to extract heme, so that the environmental pollution can be reduced, the economic benefit and the social benefit of pig blood can be increased, and the pig blood resource extraction method has high application value and good market prospect.
At present, the research on purifying heme at home and abroad is more, but the production mainly adopts the traditional glacial acetic acid method or the acidic acetone method. The glacial acetic acid method generally uses blood cell liquid or blood cell powder as raw materials, and adds glacial acetic acid and 1.5% sodium chloride according to 4-5 times volume, and then heating at about 100deg.C for 30-60min, and producing heme crystal in the course of cooling, and separating and cleaning to obtain heme with higher purity. The method has the main defects that glacial acetic acid has high boiling point and is difficult to recycle, the direct discharge environmental pollution is large, the sewage treatment cost is high, and high-purity heme is difficult to obtain by single crystallization. The acidic acetone method generally comprises precipitating hemoglobin in pig blood cells with protein denaturant such as chloroform, filtering, collecting mixed precipitate, adding acidic acetone solution with volume of about 5 times, adjusting pH to weak acidity, stirring and leaching, collecting acetone extract solution, crystallizing heme from acidic acetone solution under appropriate conditions, and cleaning to obtain pure heme product. The traditional method has the defects that the protein denaturant has certain toxicity, the dosage of acetone is large, the acetone is expensive and controlled, the acetone is easy to volatilize, the problem of environmental pollution is solved, the product cost is high, and the application is limited. Both the glacial acetic acid method and the acidic acetone method denature the hemoglobin in the process of extracting the heme, so that the value of the hemoglobin as animal source high-quality protein can be reduced or lost, pig blood resources can not be comprehensively utilized, and serious environmental pollution can be caused.
Therefore, the invention aims to provide a method for overcoming the main defects of the traditional glacial acetic acid method and the acidic acetone method, realizing the comprehensive utilization of pig hemoglobin and being suitable for large-scale preparation of high-purity heme on production. The method takes pig blood globulin powder as a raw material, adopts a composite enzymolysis method to efficiently degrade hemoglobin, separates and recovers protein peptide to obtain a precipitate enriched in heme, and prepares high-grade heme by ethanol alkali dissolution and acid precipitation purification and glacial acetic acid crystallization precipitation.
Disclosure of Invention
The invention aims to provide a method for preparing heme by enzymolysis and alcohol extraction. In addition, the ethanol is easy to recycle, can avoid chemical pollution caused by organic solvents, is safe and environment-friendly, and has simple and controllable production process.
The technical aim of the invention is realized by the following technical scheme: a method for preparing heme by enzymolysis and alcohol extraction comprises the following preparation steps:
s1: mixing pig blood globulin powder with water, regulating pH to alkaline, stirring for dissolving, adding compound proteolytic enzyme for enzymolysis, regulating pH of enzymolysis solution to acidity with acid liquor, centrifuging, and collecting precipitate; heating water for stirring and washing the precipitate, and centrifugally collecting the precipitate; adding a proper amount of water into the precipitate for suspension, and spray-drying to obtain a heme extraction crude product;
s2: adding the heme extraction crude product obtained in the step S1 into an alkaline ethanol solution to dissolve heme, centrifuging, regulating the pH of the centrifugate to be weak acid to separate out the heme, and centrifuging and collecting to obtain a heme purification precipitate;
s3: adding the heme purified precipitate obtained in the step S2 into glacial acetic acid solution, heating, cooling, crystallizing, and centrifuging to obtain heme crystal precipitate;
s4: washing the heme crystal precipitate obtained in the step S3 with water, washing with alcohol, and filtering; filtering to obtain crystal filter cake, adding purified water into the crystal filter cake, stirring to obtain suspension, and spray drying to obtain pure heme crystal.
The invention is further provided with: the heme crude product is prepared by the following method:
s1: mixing pig blood protein powder and pure water at 45-50 ℃ according to the mass-volume ratio of 1:18-25, mixing and stirring for 0.5-1h; adjusting pH to 9-10 with alkali solution, stirring and dissolving for 30-60min;
s2: adding compound protease into pig blood globulin powder dissolving solution, stirring at 50-55deg.C for enzymolysis for 1-3 hr, regulating pH value of the enzymolysis solution to 4-5 with acid solution, standing for 30min, centrifuging, and collecting heme enriched precipitate;
s3: adding pure water at 45-60 ℃ into the heme enriched precipitate, stirring and washing for 30-60min, wherein the mass ratio of the pure water to the heme enriched precipitate is 10-15:1, centrifugally collecting the precipitate, adding a proper amount of water into the precipitate, stirring the precipitate into uniform suspension, and carrying out spray drying to obtain a heme crude product.
The invention is further provided with: the alkali liquor is one of ammonia water, sodium hydroxide, potassium hydroxide and sodium carbonate solution; the acid liquid is hydrochloric acid solution.
The invention is further provided with: the mass ratio of the compound protease to the pig blood globulin powder is 0.2-0.5:100.
the invention is further provided with: the compound protease is formed by mixing animal proteolytic enzyme, alkaline protease and neutral protease.
The invention is further provided with: the mass ratio of the animal proteolytic enzyme to the alkaline protease to the neutral protease is 1:1:2.
the invention is further provided with: the heme purification precipitate is prepared by the steps of:
s1: adding the crude heme product into 85-95% ethanol solution, and stirring for 30min; the mass ratio of the heme crude product to the ethanol solution is 1:25-30;
s2: adding 4M sodium hydroxide into the mixed solution of the crude heme product obtained in the step S1 and the ethanol solution to adjust the pH of the solution to 11-12, stirring for 1-2h at 25-30 ℃, centrifuging to remove sediment, adding 2-4M hydrochloric acid into the centrifugate to adjust the pH to 5.5-6, and standing for 30-60min; and centrifuging to collect heme purified precipitate.
The invention is further provided with: the heme crystal precipitate is prepared by the steps of:
s1: adding the heme purification precipitate into a glacial acetic acid solution with the concentration of 90-95%, stirring for 0.5h, heating to 85-95 ℃, stirring for 1h, cooling to 40-50 ℃, centrifuging, and collecting to obtain heme crystal precipitate, wherein the mass ratio of the heme purification precipitate to the glacial acetic acid solution is 1:15-20.
The invention is further provided with: the washing step in S4 includes: adding 45-60 ℃ water into the heme crystal sediment obtained in the step S3, wherein the ratio of the heme crystal sediment to the water is 1: 30-50, stirring and cleaning for 1-2h, and centrifugally collecting to obtain the water-washed heme crystal.
The invention is further provided with: the alcohol washing step in S4 includes: adding absolute ethyl alcohol into the water-washed heme crystal obtained in the step S3, stirring and cleaning for 0.5-1h, wherein the mass volume ratio of the water-washed heme crystal to the absolute ethyl alcohol is 1:10-20 parts of a base; filtering to obtain heme crystal after alcohol washing.
The beneficial effects of the invention are as follows:
1. the invention provides a new heme extraction and purification method, which takes pig blood globulin powder as a raw material to efficiently degrade hemoglobin by adopting a composite enzymolysis method, separates and recovers protein peptide to obtain a heme enriched precipitate, and prepares high-grade heme by ethanol alkali dissolution and acid precipitation and purification and glacial acetic acid crystallization; according to the method, the protein peptide is recovered through enzymolysis, and the heme is extracted, so that the comprehensive utilization of the hemoglobin can be realized, the consumption of glacial acetic acid is reduced, the purity of the heme is improved, the environmental pollution caused by protein waste residues in the production process is greatly reduced, the cost of environmental protection treatment is reduced, and the production process condition is controllable.
2. The compound protease in the application adopts the mixture of animal proteolytic enzyme, alkaline protease and neutral protease, expands the range of protein types which can be acted by the compound protease, ensures that the compound protease can hydrolyze proteins at different peptide bond points under different pH conditions, is favorable for fully degrading hemoglobin, ensures that the hemoglobin is degraded into small molecular peptides which are easily dissolved in acidic aqueous solution, has small solubility in the acidic aqueous solution, and can be fully separated from the small peptides for enrichment after precipitation; the decolorized small peptide obtained by degrading and separating the compound protease has good biological activity and further development and utilization values, and the method can realize high-efficiency comprehensive utilization of the pig blood globulin.
3. The precipitate centrifuged after the pH of the composite protease enzymolysis liquid is regulated is stirred and washed by pure water at 45-60 ℃, so that protein and water-soluble impurities carried in the precipitate of heme can be dissolved and separated from the heme, the content of heme in the crude product can be improved, and the subsequent ethanol purification and glacial acetic acid crystallization are facilitated.
4. The combined use of the heme ethanol alkali dissolution acid precipitation purification method and the glacial acetic acid crystallization method in the application patent is a relatively efficient heme crystallization purification mode which is found after a large amount of long-term researches: the crude product is added into ethanol solution and then uniformly suspended in the solution in an insoluble precipitation form, after the pH value is regulated by alkali liquor, heme is dissolved in alkaline ethanol solution, protein and polypeptide components are indissoluble, the protein and polypeptide components can be removed by filtration or centrifugation, the pH value is regulated to be weak acid by hydrochloric acid, heme can be fully separated out from the solution, and the purified precipitate of heme with the content of about 50 percent is obtained. The combination of the two methods can reduce the consumption of glacial acetic acid for heme crystallization, reduce the impurity content in crystal precipitation, be favorable for crystal cleaning, improve the purity of the crystal, and ensure that ethanol is easy to recycle and the waste discharge is greatly reduced.
5. According to the method, most of protein peptide enriched heme is recovered through enzymolysis, the protein purified heme is further removed through ethanol alkali dissolution and acid precipitation, the solvent consumption is reduced for subsequent glacial acetic acid crystallization, the crystal cleaning operation is simplified, the heme content is improved, the foundation is laid, the comprehensive utilization of the hemoglobin can be realized, the production cost is greatly reduced, the high-purity heme with the content of more than 98% is prepared, and the problems of high environmental pollution and high environmental protection treatment cost in the traditional method are avoided.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a photomicrograph of a heme crystal obtained by the present invention.
FIG. 2 is a photomicrograph of a heme control crystal.
FIG. 3 is an HPLC chart of a heme crystal according to an embodiment of the present invention;
FIG. 4 is an HPLC plot of a heme control (HPLC. Gtoreq.99%, available from sigma).
Detailed Description
The technical scheme of the present invention will be clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.
Examples
1. Preparation of heme crude product
S1: mixing 1Kg pig blood globulin powder with pure water at 45-50 ℃ according to the mass-volume ratio of 1:20, mixing and stirring for 0.5-1h; adjusting pH to 10 with sodium hydroxide solution, stirring and dissolving for 30-60min;
s2: adding compound protease into pig blood protein powder dissolving liquid, wherein the mass ratio of the compound protease to the pig blood protein powder is 0.3:100, the composite protease is formed by mixing animal proteolytic enzyme, alkaline protease and neutral protease, wherein the mass ratio of the animal proteolytic enzyme to the alkaline protease to the neutral protease is 1:1:2, stirring and carrying out enzymolysis for 2 hours at 50 ℃, regulating the pH value of the enzymolysis liquid to 4.5 by using a 4M hydrochloric acid solution, standing for 30 minutes, and centrifugally collecting heme enriched precipitate;
s3: adding 50 ℃ water into the heme enriched precipitate, stirring and washing for 60min, wherein the mass ratio of the water to the heme enriched precipitate is 10:1, then centrifugally collecting the precipitate, adding a proper amount of water into the precipitate, stirring the precipitate into uniform suspension, and carrying out spray drying to obtain 0.225Kg of crude heme product, wherein the heme content is 8%.
2. Preparation of heme purification precipitate
S1: adding the heme crude product into 90% ethanol solution, and stirring for 30min; the mass ratio of the heme crude product to the ethanol solution is 1:28;
s2: adding 4M sodium hydroxide into the mixed solution of the crude heme product obtained in the step S1 and the ethanol solution to adjust the pH of the solution to 11.5, stirring for 1.5h, filtering to remove insoluble precipitate, adding 2-4M hydrochloric acid into the filtrate to adjust the pH to 6.0, and standing for 60min; the heme purified precipitate was collected by centrifugation, and the wet weight of the precipitate was 0.32Kg and the heme content was 45% (dry weight basis).
3. Preparation of heme crystal precipitate
S1: adding the heme purification precipitate into a 90% glacial acetic acid solution, stirring for 0.5h, heating to 90 ℃, stirring for 1h, cooling to 45 ℃, centrifuging, and collecting heme crystal precipitate, wherein the mass ratio of the heme purification precipitate to the glacial acetic acid solution is 1:15.
3. Heme crystal precipitation cleaning step
S1: stirring and cleaning the heme crystal sediment with water at 50 ℃ for 1h, wherein the mass-volume ratio of the heme crystal sediment to the water is 1:40, and centrifugally collecting the water-washed heme crystal.
S2: adding the washed heme crystal into absolute ethyl alcohol, stirring and cleaning for 1h, wherein the mass volume ratio of the washed crystal to the absolute ethyl alcohol is 1:15; filtering to obtain crystal filter cake, adding proper amount of water into the crystal filter cake, stirring to obtain suspension, and spray drying to obtain pure heme crystal.
Heme crystal purity determination:
the method for measuring the purity of heme crystals by referring to the national standard GB 1903.52-2021 of food nutrition enhancer methemoglobin chloride comprises the following specific steps:
1. preparing a reference substance determination solution: precisely weighing about 20mg of reference substance by a ten-thousandth balance, placing in a glass sample bottle, placing in a drying oven, drying in vacuum to constant weight at 105 ℃, dissolving in 220ml of 0.1M NaOH solution, magnetically stirring for more than 30min, transferring into a volumetric flask, cleaning a beaker with a small amount of 0.1M NaOH, fixing the volume to 250ml, shaking uniformly, taking a proper amount of solution, and filtering with a 0.45 mu M filter membrane to obtain a filtrate, namely the solution to be measured.
2. Preparation of test sample measurement solution: the preparation method is the same as that of the reference substance solution.
3. High performance liquid chromatography measurement conditions: LC-300 analytical high performance liquid chromatograph; chromatographic column: thermo Bestal C 18 (250 mm. Times.4.6 mm,5 μm); mobile phase: methanol: 1% glacial acetic acid=73: 27 (volume ratio); flow ofThe speed is 1ml/min; detection wavelength: 385nm; column temperature: 35 ℃; the sample injection amount is 20ul.
4. Calculating the content of methemoglobin chloride: according to the peak areas of the test sample and the reference substance measuring solution, calculating the heme content of the test sample by adopting a single-point appearance method, wherein the formula is as follows:
methemoglobin content = (sample peak area/control peak area × control assay mass concentration × 99%)/sample assay mass concentration × 100%.
The measurement results were as follows:
FIG. 2 is an HPLC chart of heme crystals, and Table 1 shows the results of detection of the heme crystals prepared according to the examples in 3 sets of parallel experiments and standards, with respect to the control, the purity of heme crystals is not less than 98%.
FIG. 3 is an HPLC chromatogram of a heme control with a labeled purity of 99% or more.
The result shows that the content of heme crystals prepared by the method is basically the same as that of a standard substance, but the preparation method is different from the traditional glacial acetic acid crystallization method, protein peptide is recovered through enzymolysis, and then the protein peptide is purified through ethanol alkali dissolution and acid precipitation, and the glacial acetic acid is crystallized and separated out to prepare high-grade heme, so that the method avoids chemical pollution caused by the traditional organic solvent, is safe and environment-friendly, has low comprehensive production cost and simple and controllable process.
Table 1 shows the results of the parallel experiments and the standard test of the heme crystal 3 groups prepared in the first example
Claims (10)
1. A method for preparing heme by enzymolysis and alcohol extraction is characterized in that: the preparation method comprises the following preparation steps:
s1: mixing pig blood globulin powder with water, regulating pH to alkaline, stirring for dissolving, adding compound proteolytic enzyme for enzymolysis, regulating pH of enzymolysis solution to acidity with acid liquor, centrifuging, and collecting precipitate; heating water for stirring and washing the precipitate, and centrifugally collecting the precipitate; adding a proper amount of water into the precipitate for suspension, and spray-drying to obtain a heme extraction crude product;
s2: adding the heme extraction crude product obtained in the step S1 into an alkaline ethanol solution to dissolve heme, centrifuging, regulating the pH of the centrifugate to be weak acid to separate out the heme, and centrifuging and collecting to obtain a heme purification precipitate;
s3: adding the heme purified precipitate obtained in the step S2 into glacial acetic acid solution, heating, cooling, crystallizing, and centrifuging to obtain heme crystal precipitate;
s4: washing the heme crystal precipitate obtained in the step S3 with water, washing with alcohol, and filtering; filtering to obtain crystal filter cake, adding purified water into the crystal filter cake, stirring to obtain suspension, and spray drying to obtain pure heme crystal.
2. The method for preparing heme by enzymatic alcohol extraction according to claim 1, characterized in that: the heme crude product is prepared by the following method:
s1: mixing pig blood protein powder and pure water at 45-50 ℃ according to the mass-volume ratio of 1:18-25, mixing and stirring for 0.5-1h; adjusting pH to 9-10 with alkali solution, stirring and dissolving for 30-60min;
s2: adding compound protease into pig blood globulin powder dissolving solution, stirring at 50-55deg.C for enzymolysis for 1-3 hr, regulating pH value of the enzymolysis solution to 4-5 with acid solution, standing for 30min, centrifuging, and collecting heme enriched precipitate;
s3: adding pure water at 45-60 ℃ into the heme enriched precipitate, stirring and washing for 30-60min, wherein the mass ratio of the pure water to the heme enriched precipitate is 10-15:1, centrifugally collecting the precipitate, adding a proper amount of water into the precipitate, stirring the precipitate into uniform suspension, and carrying out spray drying to obtain a heme crude product.
3. The method for preparing heme by enzymatic alcohol extraction according to claim 2, characterized in that: the alkali liquor is one of ammonia water, sodium hydroxide, potassium hydroxide and sodium carbonate solution; the acid liquid is hydrochloric acid solution.
4. The method for preparing heme by enzymatic alcohol extraction according to claim 2, characterized in that: the mass ratio of the compound protease to the pig blood globulin powder is 0.2-0.5:100.
5. the method for preparing heme by enzymatic alcohol extraction according to claim 2, characterized in that: the compound protease is formed by mixing animal proteolytic enzyme, alkaline protease and neutral protease.
6. The method for preparing heme by enzymatic alcohol extraction according to claim 5, characterized in that: the mass ratio of the animal proteolytic enzyme to the alkaline protease to the neutral protease is 1:1:2.
7. the method for preparing heme by enzymatic alcohol extraction according to claim 6, characterized in that: the heme purification precipitate is prepared by the steps of:
s1: adding the crude heme product into 85-95% ethanol solution, and stirring for 30min; the mass ratio of the heme crude product to the ethanol solution is 1:25-30;
s2: adding 4M sodium hydroxide into the mixed solution of the crude heme product obtained in the step S1 and the ethanol solution to adjust the pH of the solution to 11-12, stirring for 1-2h at 25-30 ℃, centrifuging to remove sediment, adding 2-4M hydrochloric acid into the centrifugate to adjust the pH to 5.5-6, and standing for 30-60min; and centrifuging to collect heme purified precipitate.
8. The method for preparing heme by enzymatic alcohol extraction according to claim 1, characterized in that: the heme crystal precipitate is prepared by the steps of:
s1: adding the heme purification precipitate into a glacial acetic acid solution with the concentration of 90-95%, stirring for 0.5h, heating to 85-95 ℃, stirring for 1h, cooling to 40-50 ℃, centrifuging, and collecting to obtain heme crystal precipitate, wherein the mass ratio of the heme purification precipitate to the glacial acetic acid solution is 1:15-20.
9. The method for preparing heme by enzymatic alcohol extraction according to claim 1, characterized in that: the washing step in S4 includes: adding 45-60 ℃ water into the heme crystal sediment obtained in the step S3, wherein the ratio of the heme crystal sediment to the water is 1: 30-50, stirring and cleaning for 1-2h, and centrifugally collecting to obtain the water-washed heme crystal.
10. The method for preparing heme by enzymatic alcohol extraction according to claim 1, characterized in that: the alcohol washing step in S4 includes: adding ethanol into the water-washed heme crystal obtained in the step S3, and stirring and cleaning for 0.5-1h, wherein the mass volume ratio of the water-washed heme crystal to the absolute ethanol is 1:10-20 parts of a base; filtering to obtain pure heme crystal.
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