CN104195195A - Method for extracting heme - Google Patents
Method for extracting heme Download PDFInfo
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- CN104195195A CN104195195A CN201410409254.9A CN201410409254A CN104195195A CN 104195195 A CN104195195 A CN 104195195A CN 201410409254 A CN201410409254 A CN 201410409254A CN 104195195 A CN104195195 A CN 104195195A
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Abstract
The invention discloses a method for extracting heme. The method comprises the following steps: carrying out centrifugal separation on fresh pig blood and collecting porcine blood corpuscles; after mixing the porcine blood corpuscles with water at a certain ratio, and stirring at a high speed so that membranes of the porcine blood corpuscles rupture; adjusting the temperature to 30-70 DEG C and the pH to 4.0-9.0, adding trypsase for enzymolysis for 6-24 hours; then, adjusting the pH of enzymatic hydrolysate after enzymolysis to 4.0-9.0 and heating to 50-90 DEG C, centrifugally separating the enzymatic hydrolysate and collecting sediment, wherein the sediment is a crude heme product; adding a sodium hydroxide solution to the crude heme product, heating to 50-90 DEG C, and stirring for dissolving the crude heme product; after cooling the solution, adding CMC, stirring evenly, centrifugally separating, and collecting CMC-heme; and separating the CMC with an ultrafiltration membrane and drying the heme, thus obtaining the finished product of heme. According to the method, no organic solvent is needed, thus achieving good safety and environmental friendliness; and production process is simple and controllable, and the purity is over 85%.
Description
Technical field
The present invention relates to a kind of extracting method of protoheme.
Background technology
In pig blood, contain 0.5% protoheme, the chromogenic reagent that it not only can be used as in meat product is used, and also can be used as semi-synthetic bilirubin raw material, and is anticancer specifics, also can be used as clinically benefit chalybeate, the anemia that treatment is caused by iron deficiency.The research of current domestic protoheme is more popular, but produce upper separation and Extraction, does not break through yet traditional method, obtain highly purified protoheme, and what still lean on is acid acetone precipitation.This method first, with the oxyphorase in chloroform precipitation pig blood cell, is then filtered collecting precipitation, adds the concentrated hydrochloric acid acetone soln of 5 times of volumes 2.5%, and stirring and leaching, collects acetone haemachrome solution, and last vacuum filtration reclaims acetone, obtains protoheme sterling.The shortcoming of this traditional method is complex process, uses a large amount of organic solvents, dangerous environmental protection, and the protoheme oiliness obtaining is large, needs the main equipments such as distillation tower, and equipment investment is very large, and product cost is high, and application is restricted.
Summary of the invention
The present invention is in order to solve existing protoheme extracting method complex process, uses the technical problem of a large amount of organic solvents, the invention provides a kind of extracting method of protoheme of novel environment friendly.
In order to reach above-mentioned technique effect, the present invention takes following technical scheme:
An extracting method for protoheme, comprises the following steps:
Step 1: by the centrifugation of fresh pig blood, collect pig blood cell; Fresh pig blood carries out centrifugation, rotating speed >=5000rpm through continuous separating machine.
Step 2: the separated heme peptide of enzymatic hydrolysis: after pig blood cell is mixed by a certain percentage with water, high-speed stirring makes pig blood cell rupture of membranes, then adjust the temperature to 30～70 ℃, pH4.0～9.0, add again trypsinase to carry out enzymolysis 6～24h, then regulate enzymolysis solution pH to 4.0～9.0 after enzymolysis, and be heated to 50～90 ℃ of temperature; By enzymolysis solution centrifugation collecting precipitation, this is precipitated as protoheme crude product again; This step is for being used trypsin hydrolyzing oxyphorase, oxyphorase is resolved into micromolecular peptide, the hydrophobic protoheme that is wrapped in oxyphorase inside comes out, on protoheme, be also connected with the little peptide that some are not hydrolyzed, regulate pH, heat treated postmenstruation, peptide chain sex change forms precipitation together with hydrophobic protoheme.
Step 3: heme peptide again dissolves and adsorbs: add sodium hydroxide solution and be heated to 50～90 ℃ of temperature in protoheme crude product, stir and make protoheme dissolving crude product; After solution is cooling, add Xylo-Mucine CMC, stir; CMC-protoheme crude product is carried out to centrifugation, collect and obtain CMC-protoheme; CMC is the hydrophilic substance of a kind of macromole, toughness, and it can adsorb the mashed prod that protoheme forms thickness, makes it separated with little peptide.
Step 4: the separation of CMC-protoheme: use ultra-filtration membrane separation of C MC and protoheme that molecular weight cut-off is 500～50000Da, get ultrafiltration trapped substance after ultrafiltration, dry, be protoheme finished product.
Further technical scheme is: described pig blood cell and the mass ratio of water are 1:1～3.
Further technical scheme is: described tryptic add-on is in every kilogram of pig blood cell, to add 50～5,000,000 U enzyme activity units.
Further technical scheme is: described enzymolysis solution centrifugation adopts horizontal screw centrifuge or three axle whizzers, and centrifugal rotational speed is greater than 3000rpm, more than centrifugal 20min.
Further technical scheme is: the massfraction of described sodium hydroxide solution is 0.1～5%; The add-on of sodium hydroxide solvent is 15～30 times of protoheme crude product quality.
Further technical scheme is: the final concentration that adds CMC in step 3 is 0.1～10%, adds after Xylo-Mucine CMC, and the massfraction of CMC is 0.1～10%.
Further technical scheme is: described CMC-protoheme crude product carries out centrifugation and adopts disk plate centrifuge, and rotating speed is greater than 5000rpm, and centrifugation time is 30min.
Further technical scheme is: the material of described ultra-filtration membrane is polymeric amide, and the membrane module of ultra-filtration membrane is rolling, and ultrafiltration temperature is 60 ℃, and material liquid pH is 4～10, and ultrafiltration pressure is 0.01MPa, and the ultrafiltration time is 30～60min.
The present invention compared with prior art, has following beneficial effect:
Present method desmoenzyme solution and CMC absorption method are extracted protoheme, without any need for organic solvent, and safety and environmental protection, production technique is simply controlled, and equipment investment is little, can continuous operation; The protoheme extracting is oiliness not, and purity is more than 85%.
Embodiment
Below in conjunction with embodiments of the invention, the invention will be further elaborated.
The extracting method of protoheme, comprises the following steps:
1, pig blood cell pre-treatment: fresh pig blood, through high precision continuous separating machine separated (rotating speed >=5000rpm), is collected and obtained pig blood cell.
2, the separated heme peptide of enzymatic hydrolysis: add a certain amount of blood cell in the enzymatic vessel of jacketed type, the water that adds certain mass, high-speed stirring 10min～2h, under Hyposmolality, blood cell rupture of membranes, discharge oxyphorase, enzymatic vessel temperature is elevated to 30～70 ℃ simultaneously, dilute hydrochloric acid regulates enzymolysis solution pH4.0～9.0, adds trypsinase, and add-on adds 50～5,000,000 U enzyme activity units by every 1kg blood cell, after enzymolysis 6-24h, regulate enzymolysis solution pH to 4.0～9.0, and be heated to 50～90 ℃ of maintenance 30min, processing finishes.Horizontal screw centrifuge or three axle whizzer centrifugations for enzymolysis solution, rotating speed is greater than 3000rpm, and more than centrifugal 20min, collecting precipitation.This is precipitated as protoheme crude product.
3, heme peptide again dissolves and adsorbs: in enzymatic vessel, add heme peptide precipitation, add 0.1%～5% sodium hydroxide solution of 15～30 times of quality, be heated to 50～90 ℃, rapid stirring dissolves heme peptide.After solution is cooling, add Xylo-Mucine (CMC), making its final concentration is 0.1～10%, stirs.CMC is the hydrophilic substance of a kind of macromole, toughness, and it can adsorb the mashed prod that protoheme forms thickness, makes it separated with little peptide.Disk plate centrifuge centrifugation, rotating speed is greater than 5000rpm, more than centrifugal 30min, collects CMC-protoheme.
4, CMC-protoheme and peptide is separated: use ultrafiltration apparatus separation of C MC and protoheme, membrane module is rolling, and material is polymeric amide, ultra-filtration membrane molecular weight cut-off is 500～50000Da, and ultrafiltration temperature is 60 ℃, and material liquid pH is 4～10, ultrafiltration pressure is 0.01MPa, and the loop ultrafiltration time is 40min.After ultrafiltration, get ultrafiltration trapped substance, dry, be protoheme finished product.
Embodiment 1:
In jacketed type enzymatic vessel, add the separated fresh pig blood cell 150kg obtaining, adding water 150kg mixes, rapid stirring (30 hertz of frequencies) rupture of membranes 1h, then enzymatic vessel temperature is elevated to temperature 50 C, with dilute hydrochloric acid solution, regulates pH to 7.0, then add trypsinase 4.5kg, after dissolving, stirring velocity reduces (14 hertz of frequencies), after enzymolysis 10h, regulate enzymolysis solution pH to 5.0, and be heated to 80 ℃ and maintain 30min, processing finishes.After cooling, separated with horizontal screw centrifuge, rotating speed 4000rpm, centrifugal 20min, collecting precipitation obtains protoheme crude product, and purity is 30%.
Protoheme crude product 10kg is put into enzymatic vessel, add 200kg0.5% sodium hydroxide solution and be heated to 90 ℃, rapid stirring makes its dissolving.After solution is cooling, add Xylo-Mucine 6kg, stir.Disk plate centrifuge 5000rpm centrifugation 30min, collects pasty state precipitation, i.e. CMC-protoheme.
Pasty state precipitation 10kg by collecting, adds water 40kg, and regulating pH is 9, adopts the spiral wound film device ultrafiltration that molecular weight cut-off is 1000Da, and ultrafiltration temperature is 60 ℃, and ultrafiltration pressure is 0.01MPa, and the loop ultrafiltration time is 30min.Trapped substance drying after ultrafiltration is protoheme finished product.Protoheme purity is 87% after testing.Embodiment 2:
In jacketed type enzymatic vessel, add the separated fresh pig blood cell 150kg obtaining, adding water 300kg mixes, rapid stirring (30 hertz of frequencies) rupture of membranes 1h, then enzymatic vessel temperature is elevated to 40 ℃ of temperature, with dilute hydrochloric acid solution, regulates pH to 5.0, then add trypsinase 4.5kg, after dissolving, stirring velocity reduces (14 hertz of frequencies), after enzymolysis 15h, regulate enzymolysis solution pH to 8.0, and be heated to 80 ℃ and maintain 30min, processing finishes.After cooling, separated with horizontal screw centrifuge, rotating speed 4000rpm, centrifugal 20min, collecting precipitation obtains protoheme crude product, and purity is 30%.
Protoheme crude product 10kg is put into enzymatic vessel, add 150kg2% sodium hydroxide solution and be heated to 70 ℃, rapid stirring makes its dissolving.After solution is cooling, add Xylo-Mucine 10kg, stir.Disk plate centrifuge 5000rpm centrifugation 30min, collects pasty state precipitation, i.e. CMC-protoheme.
Pasty state precipitation 10kg by collecting, adds water 40kg, and regulating pH is 9, adopts the spiral wound film device ultrafiltration that molecular weight cut-off is 5000Da, and ultrafiltration temperature is 60 ℃, and ultrafiltration pressure is 0.01MPa, and the loop ultrafiltration time is 30min.Trapped substance drying after ultrafiltration is protoheme finished product.Protoheme purity is 89% after testing.
Embodiment 3:
In jacketed type enzymatic vessel, add the separated fresh pig blood cell 150kg obtaining, adding water 200kg mixes, rapid stirring (30 hertz of frequencies) rupture of membranes 1h, then enzymatic vessel temperature is elevated to temperature 70 C, with dilute hydrochloric acid solution, regulates pH to 9.0, then add trypsinase 4.5kg, after dissolving, stirring velocity reduces (14 hertz of frequencies), after enzymolysis 15h, regulate enzymolysis solution pH to 8.0, and be heated to 90 ℃ and maintain 30min, processing finishes.After cooling, separated with horizontal screw centrifuge, rotating speed 4000rpm, centrifugal 20min, collecting precipitation obtains protoheme crude product, and purity is 29%.
Protoheme crude product 10kg is put into enzymatic vessel, add 300kg0.1% sodium hydroxide solution and be heated to 70 ℃, rapid stirring makes its dissolving.After solution is cooling, add Xylo-Mucine 20kg, stir.Disk plate centrifuge 5000rpm centrifugation 30min, collects pasty state precipitation, i.e. CMC-protoheme.
Pasty state precipitation 10kg by collecting, adds water 40kg, and regulating pH is 9, adopts the spiral wound film device ultrafiltration that molecular weight cut-off is 20000Da, and ultrafiltration temperature is 60 ℃, and ultrafiltration pressure is 0.01MPa, and the loop ultrafiltration time is 30min.Trapped substance drying after ultrafiltration is protoheme finished product.Protoheme purity is 88% after testing.
Although with reference to explanatory embodiment of the present invention, invention has been described here, above-described embodiment is only preferably embodiment of the present invention, embodiments of the present invention are not restricted to the described embodiments, should be appreciated that, those skilled in the art can design a lot of other modification and embodiments, and these are revised and within embodiment will drop on the disclosed principle scope and spirit of the application.
Claims (8)
1. an extracting method for protoheme, is characterized in that comprising the following steps:
Step 1: by the centrifugation of fresh pig blood, collect pig blood cell;
Step 2: the separated heme peptide of enzymatic hydrolysis: after pig blood cell is mixed by a certain percentage with water, high-speed stirring makes pig blood cell rupture of membranes, then adjust the temperature to 30～70 ℃, pH4.0～9.0, add again trypsinase to carry out enzymolysis 6～24h, then regulate enzymolysis solution pH to 4.0～9.0 after enzymolysis, and be heated to 50～90 ℃ of temperature; By enzymolysis solution centrifugation collecting precipitation, this is precipitated as protoheme crude product again;
Step 3: heme peptide again dissolves and adsorbs: add sodium hydroxide solution and be heated to 50～90 ℃ of temperature in protoheme crude product, stir and make protoheme dissolving crude product; After solution is cooling, add Xylo-Mucine CMC, stir; CMC-protoheme crude product is carried out to centrifugation, collect and obtain CMC-protoheme;
Step 4: the separation of CMC-protoheme: use ultra-filtration membrane separation of C MC and protoheme that molecular weight cut-off is 500～50000Da, get ultrafiltration trapped substance after ultrafiltration, dry, be protoheme finished product.
2. the extracting method of a kind of protoheme according to claim 1, is characterized in that described pig blood cell and the mass ratio of water are 1:1～3.
3. the extracting method of a kind of protoheme according to claim 1, is characterized in that described tryptic add-on is in every kilogram of pig blood cell, to add 50～5,000,000 U enzyme activity units.
4. the extracting method of a kind of protoheme according to claim 1, is characterized in that described enzymolysis solution centrifugation adopts horizontal screw centrifuge or three axle whizzers, and centrifugal rotational speed is greater than 3000rpm, more than centrifugal 20min.
5. the extracting method of a kind of protoheme according to claim 1, is characterized in that the massfraction of described sodium hydroxide solution is 0.1～5%; The add-on of sodium hydroxide solvent is 15～30 times of protoheme crude product quality.
6. the extracting method of a kind of protoheme according to claim 1, is characterized in that in step 3, adding the final concentration of CMC is 0.1～10%.
7. the extracting method of a kind of protoheme according to claim 1, is characterized in that described CMC-protoheme crude product carries out centrifugation and adopts disk plate centrifuge, and rotating speed is greater than 5000rpm, and centrifugation time is 30min.
8. the extracting method of a kind of protoheme according to claim 1, is characterized in that the material of described ultra-filtration membrane is polymeric amide, and the membrane module of ultra-filtration membrane is rolling, ultrafiltration temperature is 60 ℃, material liquid pH is 4～10, and ultrafiltration pressure is 0.01MPa, and the ultrafiltration time is 30～60min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110150304A (en) * | 2019-05-10 | 2019-08-23 | 海盐县凌特生物科技有限公司 | The plant growth regulator of chloride containing ferroheme |
| CN110973412A (en) * | 2019-11-25 | 2020-04-10 | 西南民族大学 | Sea-buckthorn-flavored iron-function-enhanced beverage and preparation method thereof |
| CN113632876A (en) * | 2021-08-11 | 2021-11-12 | 山西大学 | Production process for adding artificial meat into colored wheat bran |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103030645A (en) * | 2012-12-21 | 2013-04-10 | 上海杰隆生物制品股份有限公司 | Method for preparing high-purity heme on large scale and application of heme |
| CN103060398A (en) * | 2012-12-31 | 2013-04-24 | 得利斯集团有限公司 | Extracting method of hemes |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103030645A (en) * | 2012-12-21 | 2013-04-10 | 上海杰隆生物制品股份有限公司 | Method for preparing high-purity heme on large scale and application of heme |
| CN103060398A (en) * | 2012-12-31 | 2013-04-24 | 得利斯集团有限公司 | Extracting method of hemes |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110150304A (en) * | 2019-05-10 | 2019-08-23 | 海盐县凌特生物科技有限公司 | The plant growth regulator of chloride containing ferroheme |
| CN110973412A (en) * | 2019-11-25 | 2020-04-10 | 西南民族大学 | Sea-buckthorn-flavored iron-function-enhanced beverage and preparation method thereof |
| CN113632876A (en) * | 2021-08-11 | 2021-11-12 | 山西大学 | Production process for adding artificial meat into colored wheat bran |
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Effective date of registration: 20210106 Address after: No.35, south 2nd Road, Chengdu Economic and Technological Development Zone, Sichuan 610000 Patentee after: CHENGDU TIANYI BIOLOGICAL TECHNOLOGY Co.,Ltd. Address before: 610000 group 1, Changzhen village, Yangma Town, Chongzhou City, Chengdu City, Sichuan Province Patentee before: BEIJING HONG'AN BIOLOGICAL TECHNOLOGY Co.,Ltd. |