CN117886826A - Method for extracting methemoglobin by heating and pressurizing - Google Patents
Method for extracting methemoglobin by heating and pressurizing Download PDFInfo
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- 108010061951 Methemoglobin Proteins 0.000 title claims abstract description 43
- 238000010438 heat treatment Methods 0.000 title claims abstract description 31
- 239000000843 powder Substances 0.000 claims abstract description 45
- 150000003278 haem Chemical class 0.000 claims abstract description 32
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims abstract description 23
- 108060003196 globin Proteins 0.000 claims abstract description 21
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 14
- 210000000601 blood cell Anatomy 0.000 claims abstract description 13
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- 238000007796 conventional method Methods 0.000 claims abstract 2
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- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 36
- 229910052742 iron Inorganic materials 0.000 claims description 18
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- 229960001716 benzalkonium Drugs 0.000 claims description 4
- JBIROUFYLSSYDX-UHFFFAOYSA-M benzododecinium chloride Chemical compound [Cl-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 JBIROUFYLSSYDX-UHFFFAOYSA-M 0.000 claims description 4
- 150000002191 fatty alcohols Chemical class 0.000 claims description 4
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 4
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 4
- 230000000694 effects Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims 1
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 14
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- 238000005119 centrifugation Methods 0.000 description 4
- 239000012362 glacial acetic acid Substances 0.000 description 4
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 4
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- GHXZTYHSJHQHIJ-UHFFFAOYSA-N Chlorhexidine Chemical compound C=1C=C(Cl)C=CC=1NC(N)=NC(N)=NCCCCCCN=C(N)N=C(N)NC1=CC=C(Cl)C=C1 GHXZTYHSJHQHIJ-UHFFFAOYSA-N 0.000 description 1
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a method for extracting methemoglobin chloride by heating and pressurizing, which relates to the technical field of biochemistry and comprises the following steps: the raw material is enzymolysis powder prepared from animal blood cells; adding secondary reverse osmosis water and urea into the enzymolysis powder to obtain a dissolving solution: adding a surfactant to obtain an active solution; adding hydrochloric acid into the active solution to adjust the pH value; heating and pressurizing the solution to split heme and globin; cooling the solution to separate out heme crystal, and preparing methemoglobin chloride by a conventional method; the raw materials are enzymolysis powder, so that the consumption of urea and surfactant and the heat energy required by heating and pressurizing are greatly reduced, the production cost is reduced, and the environmental pollution is reduced; the filtered permeate after the enzymolysis of blood cells can be ultrafiltered, concentrated and spray dried to produce blood peptide; after the temperature and the pressure of the solution are raised, the heme and the globin are completely resolved, so that the yield and the purity of the methemoglobin chloride are improved; through the heat preservation and pressure maintaining in a short time, the pressure is quickly released and cooled, and the occurrence of coprecipitation of precipitated heme and globin can be avoided.
Description
Technical Field
The invention relates to the technical field of biochemistry, in particular to a method for extracting hemin by utilizing enzymolysis powder of animal blood to raise temperature and pressurize.
Background
Heme chloride is used as a food nutrition enhancer for supplementing iron, listed in GB14880, and implemented in 2022, 3 and 7 days in national food safety standard of food nutrition enhancer Heme chloride, and has wider application prospect. In the production, three methods of glacial acetic acid method, enzymolysis and acetic acid combination method and acid acetone method are generally adopted to extract methemoglobin, but a large amount of organic solvent glacial acetic acid or acetone is used in the extraction process, so that the risks of environmental pollution, personal injury of operators and even fire are easily caused. Therefore, avoiding or using only a small amount of organic solvents such as acetic acid or acetone in the production process of methemoglobin chloride is a research direction with application value.
At present, in the method for extracting methemoglobin by avoiding using or using only a small amount of organic solvents such as acetic acid or acetone, the problems of unstable process, lower product purity, easy coprecipitation of heme and globin and separation exist, and the method is specifically expressed as follows:
1. patent ZL87101834 "separation and purification of hemin", avoids the use of acetic acid or acetone, catalyzed by benzyldodecyldimethylammonium chloride or other surfactants at pH 0.5-2.5, at a temperature of about 90 ℃ to boiling for extraction. But needs to be heated and cooled quickly, products cannot be produced by a little careless, the process is very unstable, and the practical application is not realized;
2. patent ZL2006100800229 "method for extracting chlorhexidine with surfactant", comprises diluting hemoglobin solution, adding fatty alcohol polyoxyethylene ether and other surfactant as catalyst, adjusting to acidity, and heating to less than 85deg.C. However, the temperature is lower, the heme and the globin are not completely resolved, and the purity of the product is lower than 95%; and only the surfactant is used for assisting dissolution, but urea is not used for dissolving the globin, and heme is easy to coprecipitate with the globin and cannot be separated;
3. the patent ZL200810120963X 'production method of heme and decolored hemoglobin powder', firstly, performing enzymolysis and drying to obtain heme semi-finished products; adding water, concentrated hydrochloric acid and nonionic surfactant, stirring, heating to 70-95 deg.C, centrifuging, collecting heme crystal, washing with water, and drying to obtain heme. As with the patent ZL2006100800229 described above, there are two drawbacks: (1) the temperature is lower, the heme and the globin are not completely resolved, so that the purity of the product is lower, about 88-92%; (2) only the surfactant is used for assisting dissolution, and urea is not used for dissolving the globin, so that heme is easy to coprecipitate with the globin and can not be separated;
4. in the patent of application No. 2017103732471, "methemoglobin and preparation method thereof", 5-7 mol/L high-concentration urea solution is adopted to dissolve blood powder, fatty alcohol polyoxyethylene ether or other surfactants are added after the blood powder is regulated to be acidic, and the temperature is heated to 90-100 ℃ to precipitate heme. However, the method has the disadvantages of large urea consumption, high production cost and environmental pollution; the method does not show the purity of the product, and through repeated experiments, the product is heated to 90-100 ℃ only, the temperature is lower, and the separation of heme and globin is incomplete, so that the purity is low.
In view of the above, a method for extracting methemoglobin chloride is needed, which solves the problems of low product purity, easy separation of heme and globin due to coprecipitation, high production cost and environmental pollution in the existing extraction process.
Disclosure of Invention
The invention aims at: in order to solve the technical problems, the invention provides a method for extracting methemoglobin by heating and pressurizing, which can ensure that heme and globin are easy to split completely, so that the extracted methemoglobin has high yield and high purity, and simultaneously reduces the production cost and environmental pollution.
The invention adopts the following technical scheme for realizing the purposes: a method for extracting methemoglobin by heating and pressurizing comprises the following steps:
s1, selecting raw materials: enzymolysis powder prepared from animal blood cells;
s2, dissolving and dispersing enzymolysis powder: adding secondary reverse osmosis water into the enzymolysis powder, stirring and dissolving, and adding urea to accelerate dissolution to obtain a dissolving solution;
s3, improving activity: adding a surfactant or a mixture of surfactants into the dissolving solution to obtain an active solution;
s4, adjusting the pH value: hydrochloric acid is added into the active solution to adjust the pH value to 2.0-3.0;
s5, heating and pressurizing: pumping the solution with the pH value regulated into a pressure vessel, heating by steam to 140-150 ℃, maintaining the pressure at 0.36-0.48 MPa, and preserving the heat and the pressure for 10-20 minutes to split heme and globin;
s6, cooling and separating out: quick-releasing the solution, discharging, naturally cooling, precipitating heme crystal, centrifuging, washing with acetic acid solution, washing with water, drying, and pulverizing to obtain methemoglobin chloride.
Preferably, the enzymolysis powder in the step S1 is specifically obtained by spray drying concentrated solution obtained by hemolysis, protease enzymolysis, ceramic membrane filtration and filtration of animal blood cells.
Preferably, the iron content of the enzymolysis powder in S1 is 2-3%, and the purity of the methemoglobin chloride is 23-35%.
Preferably, the weight of the second-stage reverse osmosis water in the S2 is 20-40 times of the weight of the enzymolysis powder, and the added urea accounts for 2-4% of the total weight.
Preferably, the surfactant or the mixture of the surfactants in the S3 is one or a mixture of more of tween-20, benzyl dodecyl dimethyl ammonium chloride and fatty alcohol polyoxyethylene ether, and the concentration of the solution is 0.8-2.6%.
Preferably, the solution in which the heme crystals are precipitated in the S6 is subjected to centrifugation, acetic acid liquid washing, water washing, drying and crushing to obtain the methemoglobin chloride with the iron content of more than 8.42% and the purity of more than 98%.
The beneficial effects of the invention are as follows:
1. in the national food safety standard of food nutrition enhancer methemoglobin chloride, methemoglobin chloride is prepared by adopting an glacial acetic acid method, enzymolysis and acetic acid combination method, and is prepared by adopting an acidic acetone method in actual production, and the technical method of the application only uses a small amount of acetic acid liquid to wash the centrifuged precipitate, so that a large amount of organic solvents such as glacial acetic acid or acetone and the like are avoided in the production process;
2. according to the method, fresh blood cells or blood powder and blood cell powder are not used as raw materials, the iron content after enzymolysis of protease is up to 2.0-3.0%, and the enzymolysis powder with the purity of methemoglobin reaching 23-35% is used as a raw material, so that the consumption of urea and surfactant is reduced by tens times or even hundreds times, the heat energy required by heating and pressurizing is reduced geometrically, and the production cost and environmental pollution are greatly reduced; the filtered permeate after the enzymolysis of blood cells can be subjected to ultrafiltration concentration and spray drying to produce blood peptide for comprehensive utilization;
3. hemoglobin in animal blood is connected by a covalent bond of globin and heme, and the extraction of high-purity heme is completed by opening the covalent bond, so that the separation of heme and globin is a key point of the whole technology. The covalent bond is opened under severe reaction conditions, so that the temperature is raised to 140-150 ℃ and the pressure is 0.36-0.48 MPa, and the separation is complete under the conditions of higher temperature and pressure, the yield is high, and the purity is high; while the condition in other prior art studies is 80 ℃ to boiling, resolution is incomplete, and many peptide fragments of globin remain attached to heme molecules;
4. the solution with the pH value adjusted is heated to 140-150 ℃ and the pressure is 0.36-0.48 MPa, so that the split globin is easy to coprecipitate with the precipitated heme and cannot be separated, and the failure is caused. But urea is added to dissolve split globin, and the surfactant has an auxiliary dissolving effect on the globin; the heat preservation and pressure maintaining time is shortened to 10-20 minutes, the pressure is immediately released for discharging, and the material is naturally cooled, so that the coprecipitation can be avoided;
5. the method of the technology adopts conventional speed to heat and cool, and does not need special equipment, so that the method is suitable for large-scale production.
Detailed Description
The present invention will be described in detail with reference to specific embodiments, illustrative embodiments and descriptions herein are intended to illustrate the invention, but not to limit the invention.
Example 1
S1, 1 ton of yak blood cells, carrying out conventional hemolysis, protease enzymolysis and ceramic membrane filtration, and spray drying a concentrated solution to obtain 26kg of enzymolysis powder, wherein the enzymolysis powder is used as a raw material for heating and pressurizing extraction, the iron content of the enzymolysis powder is 2.82%, and the purity of methemoglobin chloride is 32.83%.
S2, adding 40 times of secondary reverse osmosis water (1040L) into the enzymolysis powder, stirring and dissolving, adding 43kg of urea (4%), 28kg of Tween-20 (2.6%), stirring and dissolving, regulating the pH to 2.0 by using hydrochloric acid, pumping into a pressure container, heating by steam to 150 ℃, keeping the pressure at 0.48MPa, keeping the temperature for 10 minutes, immediately releasing pressure and discharging, and naturally cooling.
S3, cooling, separating out heme crystals, and carrying out conventional centrifugation, 20% acetic acid liquid washing, water washing, drying and crushing to obtain 5.8kg of methemoglobin chloride with the iron content of 8.54% and the purity of 99.42%.
Example 2
S1, 1 ton of cattle blood cells, carrying out conventional hemolysis, protease enzymolysis, ceramic membrane filtration and spray drying of concentrated solution to obtain 24kg of enzymolysis powder, wherein the enzymolysis powder is used as a raw material for heating and pressurizing extraction, the iron content of the enzymolysis powder is 2.23%, and the purity of methemoglobin chloride is 25.96%.
S2, adding 30 times of second-stage reverse osmosis water (720L) into enzymolysis powder, stirring and dissolving, adding 22kg of urea (3%), 15kg of fatty alcohol-polyoxyethylene ether (2%), stirring and dissolving, regulating pH to 2.6 with hydrochloric acid, pumping into a pressure container, heating by steam to 145 ℃, keeping the pressure at 0.42MPa, keeping the temperature for 15 minutes, immediately releasing pressure and discharging, and naturally cooling.
S3, cooling, separating out heme crystals, and carrying out conventional centrifugation, washing with 15% acetic acid solution, washing with water, drying and crushing to obtain 4.2kg of methemoglobin chloride with iron content of 8.45% and purity of 98.37%.
Example 3
S1, pig blood balls are 1 ton, and through conventional hemolysis, protease enzymolysis, ceramic membrane filtration and concentrated solution spray drying, 22kg of enzymolysis powder is obtained, and the enzymolysis powder is used as raw materials for heating and pressurizing extraction, wherein the iron content of the enzymolysis powder is 2.04%, and the purity of methemoglobin chloride is 23.75%.
S2, adding 20 times of secondary reverse osmosis water (440L) into enzymolysis powder, stirring and dissolving, adding 9.2kg of urea (2%), mixing 3.7kg of benzyl dodecyl dimethyl ammonium chloride and tween-20 (0.4% each according to the proportion of 1:1), stirring and dissolving, regulating the pH value to 3.0 by using hydrochloric acid, pumping into a pressure container, heating by steam to 140 ℃, keeping the pressure at 0.36MPa, keeping the temperature and the pressure for 20 minutes, immediately releasing pressure and discharging, and naturally cooling.
S3, cooling, separating out heme crystals, and performing conventional centrifugation, 10% acetic acid liquid washing, water washing, drying and crushing to obtain 3.5kg of methemoglobin chloride with the iron content of 8.43% and the purity of 98.14%.
Comparative example 1
According to the method for extracting methemoglobin, 1 ton of pig blood is treated to obtain 22kg of enzymolysis powder as raw material, other conditions are unchanged, but steam is heated to about 125 ℃, the pressure is about 0.23MPa, and the temperature and pressure are maintained for 20 minutes; after the subsequent treatment, 4.1kg of methemoglobin chloride with an iron content of 7.71% and a purity of 89.76% was obtained.
Comparison table for result difference caused by different heating and pressurizing values
It can be seen that the iron content and purity of the product are lower when the temperature and pressure are higher and lower by conventional equipment.
While patent 202222213394.1 "a continuous flow reactor for continuously extracting hemin", which rapidly heats the reaction solution from room temperature to 120-130 ℃ within 2-4 minutes, requires special equipment; nor are the iron content, purity and yield of the hemin produced.
Comparative example 2
According to the method for extracting methemoglobin, 1 ton of pig blood is treated to obtain 22kg of enzymolysis powder as raw material, other conditions are unchanged, but 22kg of sprayed pig blood protein powder is used for replacing the enzymolysis powder, steam is heated to 140 ℃, the pressure is 0.36MPa, and the temperature and pressure are maintained for 20 minutes; after the subsequent treatment, 0.55kg of methemoglobin chloride with an iron content of only 3.34% and a purity of 38.88% is obtained.
Comparison table of difference of results caused by replacement of enzymolysis powder by porcine hemoglobin powder
Therefore, the iron content and purity of the product are greatly reduced by using the hemocyte protein powder to replace the enzymolysis powder, and the quality of the product is far from the national standard of methemoglobin chloride.
Because the iron content of the enzymolysis powder reaches 2.0-3.0%, heme is primarily enriched, and the subsequent treatment is facilitated.
As can be seen from comparative examples 1 and 2, the enzymatic hydrolysate powder is used as a raw material, is heated and pressurized to 140-150 ℃, has the pressure of 0.36-0.48 MPa, is kept at a temperature and a pressure for a short time, and is the optimal process for extracting methemoglobin chloride.
Comparative example 3
According to the method for extracting methemoglobin, only 8.8-35.2 kg of urea is needed for treating 1 ton of pig blood balls, and 900-1800 kg of urea is needed for treating the pig blood balls and is 51-102 times of the pig blood balls according to the method of the patent with the application number of 2017103732471. Similarly, 1 ton of pig blood cells only produce 22kg of enzymolysis powder after enzymolysis, namely geometrically reduce the consumption of surfactant, geometrically reduce the heat energy required by heating, and the protease has low price, and the filtered permeate after the enzymolysis of the blood cells can be subjected to ultrafiltration concentration and spray drying to produce blood peptide for comprehensive utilization; thereby greatly reducing the production cost.
Urea usage comparison table of the process of the present application and the process of patent application No. 2017103732471:
urea dosage comparison table for two methods
While the foregoing has been provided by way of example and comparative example, the principles and embodiments of the present invention have been described in detail with reference to specific examples, and the description of the foregoing examples is only intended to facilitate the understanding of the principles of the examples, and in light of the above teachings, those skilled in the art should not be construed to limit the invention to any particular embodiment or application range depending on the examples of the invention.
Claims (8)
1. A method for extracting methemoglobin by heating and pressurizing comprises the following steps:
s1, raw materials: enzymolysis powder prepared from animal blood cells;
s2, dissolving and dispersing enzymolysis powder: sequentially adding second-stage reverse osmosis water and urea into the enzymolysis powder to dissolve so as to obtain a dissolving solution;
s3, improving activity: adding a surfactant or a mixture of surfactants into the dissolving solution to obtain an active solution;
s4, adjusting pH: adding hydrochloric acid into the active solution to adjust the pH value;
s5, heating and pressurizing: heating the solution with the pH value regulated, and then preserving heat and pressure to split heme and globin;
s6, cooling and separating out: and (3) rapidly releasing the solution, discharging, cooling, separating out heme crystals, and preparing methemoglobin chloride by a conventional method.
2. The method for extracting methemoglobin by heating and pressurizing according to claim 1, wherein the method comprises the following steps: the enzymolysis powder in the S1 is specifically obtained by spray drying concentrated solution obtained by hemolysis, protease enzymolysis, ceramic membrane filtration and filtration of animal blood cells.
3. The method for extracting methemoglobin by heating and pressurizing according to claim 1, wherein the method comprises the following steps: the iron content of the enzymolysis powder in the S1 is 2-3%, and the purity of the methemoglobin chloride is 23-35%.
4. The method for extracting methemoglobin by heating and pressurizing according to claim 1, wherein the method comprises the following steps: the weight of the second-stage reverse osmosis water in the S2 is 20-40 times of that of the enzymolysis powder; the added urea accounts for 2 to 4 percent of the total weight.
5. The method for extracting methemoglobin by heating and pressurizing according to claim 1, wherein the method comprises the following steps: one or a mixture of a plurality of surfactants in the S3 is one or a mixture of a plurality of tween-20, benzyl dodecyl dimethyl ammonium chloride and fatty alcohol polyoxyethylene ether, and the concentration of the solution is 0.8-2.6 percent.
6. The method for extracting methemoglobin by heating and pressurizing according to claim 1, wherein the method comprises the following steps: and in the step S4, the pH value is regulated to 2.0-3.0.
7. The method for extracting methemoglobin by heating and pressurizing according to claim 1, wherein the method comprises the following steps: pumping the solution with the pH value regulated in the step S5 into a pressure vessel, heating by steam to 140-150 ℃, maintaining the pressure at 0.36-0.48 MPa, and preserving the heat and the pressure for 10-20 minutes.
8. The method for extracting methemoglobin by heating and pressurizing according to claim 1, wherein the method comprises the following steps: and (3) centrifuging, washing with acetic acid solution, washing with water, drying and crushing the solution after S6 heme crystal precipitation to obtain methemoglobin chloride with iron content of more than 8.4% and purity of more than 98%.
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