CN105567759A - Method for preparing anhydrous chlorhematin by utilizing yak blood - Google Patents
Method for preparing anhydrous chlorhematin by utilizing yak blood Download PDFInfo
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- CN105567759A CN105567759A CN201610117183.4A CN201610117183A CN105567759A CN 105567759 A CN105567759 A CN 105567759A CN 201610117183 A CN201610117183 A CN 201610117183A CN 105567759 A CN105567759 A CN 105567759A
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Abstract
The invention belongs to the technical field of biology, and relates to a method for preparing anhydrous chlorhematin by utilizing yak blood. The method is characterized in that the method is carried out according to the steps of collection of erythrocyte, crushing of the erythrocyte, enzymolysis of hemoglobin and extraction of chlorhematin. According to the method disclosed by the invention, the anhydrous chlorhematin is prepared by utilizing the yak blood, so that an animal blood resource is fully and valuably utilized; the method for preparing the anhydrous chlorhematin is simple and is easy to operate, used chemical reagents are less, the cost is lower, the environment pollution is lower, and the method is convenient for industrial production.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of preparation method of anhydrous chlorides of rase protoheme, particularly a kind of method utilizing yak blood to prepare anhydrous chlorides of rase protoheme.
Background technology
Yak is one of cattle kind of China, is the sociales domestic animal in Qinghai-Tibet pastoral area and work as Domestic Livestock, has the vitality of tanacity.China is the source region of world yak, and the yak in the whole world 90% lives in 6 provinces and regions that China is Qinghai-Tibet and adjoin.Carnis Bovis grunniens protein is up to 21%, and fat is low, and amino acid kind is complete, and quality is good, raciness.Yak milk is dense thick, dry matter content about 18%, lipid content about 7%, protein content about 5%.Fur is characteristic, and Development volue is large.According to measuring and calculating, carry out comprehensive exploitation to Carnis Bovis grunniens, breast, hair, skin, bone, blood etc., can obtain the economic value added exceeding raw material 5 times, only Industry of Yaks can realize the output value of hundreds of hundred million.At present, the utilization for Carnis Bovis grunniens, breast is relatively more abundant, and yak blood is because there being the larger blood smell, and utilization ratio is not high.But in yak blood, content of hemoglobin is higher (73.34 ~ 99.48g/L), apparently higher than other animals, thus it prepares the good raw material of protohemine.
Protoheme is a kind of iron porphyrin compound, and it is the prothetic group of oxyphorase, myohaemoglobin, cytopigment, catalase and peroxidase.Mainly be present in animal blood and muscle, be the natural pigment in animal blood, be widely used in medicine, food, chemical industry, healthcare products, building and cosmetic industry.Protoheme is except can being used as the pigment additive in food, still the good a kind of iron supplementary of hypoferric anemia (IDA) curative effect is treated at present, have bioavailability high, without the advantages such as untoward reaction such as iron accumulate poisoning and gastrointestinal irritation in body, simultaneously or the important source material of anti-anemia action and antitumor drug, China is the haematoporphyrin of basic raw material in official approval in 1998 with protoheme is anti-cancer agent.U.S. FDA uses in the protohemine of official approval in July nineteen eighty-three Abbott as medicine.
At present, the preparation method about protohemine has a lot, but substantially all will use a large amount of chemical reagent, and the pollution of the waste and environment that so both cause reagent also makes production cost very high simultaneously.The present invention is intended to take yak blood as raw material, and the blood that need not add antithrombotics prepares protohemine by enzymolysis process, not only reduces production cost but also be convenient to suitability for industrialized production.
Summary of the invention
In view of above-mentioned yak blood utilization ratio is low, existing method waste reagent, contaminate environment, the defect that production cost is high preparing protohemine, the object of this invention is to provide and a kind ofly use the yak blood that utilizes that chemical reagent is few, cost is lower, environmental pollution is lower to prepare the method for anhydrous chlorides of rase protoheme.
The object of the invention is to press that surface technology scheme realizes, a kind of method utilizing yak blood to prepare anhydrous chlorides of rase protoheme, it is characterized in that carrying out, described in specific as follows according to the enzymolysis of erythrocytic collection, erythrocytic fragmentation, oxyphorase and the extraction step of protohemine:
1. erythrocytic collection
Collect fresh yak blood 20ml with 50ml test tube, filter with gauze, remove larger impurity, collect filtered liquid, leave standstill after 1 hour after blood coagulation layering, suck the flaxen liquid in upper strata, obtain lower floor red corpuscle coagulum 12ml.
2. erythrocytic fragmentation
In red corpuscle coagulum, add the deionized water of 0.5 ~ 1.25 times, put into ultrasonic wave and carry out broken 15min, stir with glass stick simultaneously, solid coagulum is fully disperseed, makes erythroclasis simultaneously, release oxyphorase; Solution is poured into fast in six small test tubes, under 60 DEG C of lucifuge conditions, heat 30min, use 3000r/min centrifuge 10min afterwards, the supernatant liquid in six small test tubes is poured in 20ml Erlenmeyer flask, remove the foreign protein of lower floor's sex change, obtain hemoglobin solutions.
3. the enzymolysis of oxyphorase
The HCl0.1mol/L standardized solution of using of hemoglobin solutions is regulated PH=6.8 ~ 7.0, and be put in 50 DEG C of thermostat water baths and heat 20min, adding enzyme activity is 4000u/g neutral protease 0.3g, temperature 45 C; Be hydrolyzed that to add enzyme activity after 5 hours be 2000u/g food flavor enzyme 0.5g, be adjusted to pH=6.8 ~ 7.0, temperature 53 DEG C; After hydrolysis reaction completes, heated and boiled is gone out enzyme.
4. the extraction of protohemine
Oxyphorase is after enzymolysis, peptide chain is disconnected, and has protoheme in hydrolyzed solution, heme peptide and peptide fragment, after adjusting pH=4.5 with HCl0.1mol/L solution, add the sodium acetate solution of mass concentration 1g/100mL, separate out precipitation, collecting precipitation with the centrifugal 10min of 3000r/min rotating speed, use the acetic acid solution of ionized water, volume fraction 50%, the washing of volume fraction 95% ethanolic soln successively once, use washed with diethylether twice again, subzero 20 DEG C of lyophilizes 8 hours, obtain protohemine.
The present invention utilizes yak blood to prepare anhydrous chlorides of rase protoheme, makes animal blood resource obtain the utilization fully having valency.The method preparing anhydrous chlorides of rase protoheme is simple, and easy handling, chemical reagent used is few, cost is lower, environmental pollution is lower, is convenient to suitability for industrialized production.
Embodiment
Embodiment 1;
1. erythrocytic collection: collect fresh yak blood 20ml with 50ml test tube, filter with gauze, removes larger impurity, collect filtered liquid, leave standstill after 1 hour after blood coagulation layering, suck the flaxen liquid in upper strata, obtain lower floor red corpuscle coagulum 12ml.
2. erythrocytic fragmentation: the deionized water adding 6ml in red corpuscle coagulum, puts into ultrasonic wave and carries out broken 15min, stirs with glass stick simultaneously, solid coagulum is fully disperseed, makes erythroclasis simultaneously, release oxyphorase.Solution is poured into fast in six small test tubes, under 60 DEG C of lucifuge conditions, heat 30min, use 3000r/min centrifuge 10min afterwards, the supernatant liquid in six small test tubes is poured in 20ml Erlenmeyer flask, remove the foreign protein of lower floor's sex change, obtain hemoglobin solutions.
3. the enzymolysis of oxyphorase: the HCl0.1mol/L standardized solution of using collecting the hemoglobin solutions obtained is regulated PH=6.8, and be put in 50 DEG C of thermostat water baths and heat 20min, adding enzyme activity is 4000u/g neutral protease 0.3g, temperature 45 C; Be hydrolyzed that to add enzyme activity after 5 hours be 2000u/g food flavor enzyme 0.5g, be adjusted to pH=6.9, temperature 53 DEG C; After hydrolysis reaction completes, heated and boiled is gone out enzyme.
4. the extraction of protohemine: oxyphorase is after enzymolysis, peptide chain is disconnected, protoheme is had in hydrolyzed solution, heme peptide and peptide fragment, after adjusting pH=4.5 with HCl0.1mol/L solution, add the sodium acetate solution of mass concentration 1g/100mL, precipitation is separated out with the centrifugal 10min of 3000r/min rotating speed, collecting precipitation, use the acetic acid solution of ionized water, volume fraction 50%, the washing of volume fraction 95% ethanolic soln successively once, use washed with diethylether twice again, subzero 20 DEG C of lyophilizes 6 hours, obtain protohemine.
Embodiment 2;
1. erythrocytic collection: with embodiment 1.
2. erythrocytic fragmentation: the deionized water adding 12ml in red corpuscle coagulum, remaining same embodiment 1.
The enzymolysis of 3 oxyphorases: the HCl0.1mol/L standardized solution of using collecting the hemoglobin solutions obtained is regulated PH=7.0, and be put in 50 DEG C of thermostat water baths and heat 20min, adding enzyme activity is 4000u/g neutral protease 0.3g, temperature 45 C; Be hydrolyzed that to add enzyme activity after 5 hours be 2000u/g food flavor enzyme 0.5g, be adjusted to pH=6.9, remaining same embodiment 1.
4. the extraction of protohemine: with embodiment 1.
Embodiment 3;
1. erythrocytic collection: with embodiment 1.
2. erythrocytic fragmentation: the deionized water adding 15ml in red corpuscle coagulum, remaining same embodiment 1.
The enzymolysis of 3 oxyphorases: the HCl0.1mol/L standardized solution of using collecting the hemoglobin solutions obtained is regulated PH=7.0, and be put in 50 DEG C of thermostat water baths and heat 20min, adding enzyme activity is 4000u/g neutral protease 0.3g, temperature 45 C; Be hydrolyzed that to add enzyme activity after 5 hours be 2000u/g food flavor enzyme 0.5g, be adjusted to pH=7.0, remaining same embodiment 1.
4. the extraction of protohemine: with embodiment 1.
Embodiment 4;
1. erythrocytic collection: with embodiment 1.
2. erythrocytic fragmentation: the deionized water adding 10ml in red corpuscle coagulum, remaining same embodiment 1.
The enzymolysis of 3 oxyphorases: the HCl0.1mol/L standardized solution of using collecting the hemoglobin solutions obtained is regulated PH=6.8, and be put in 50 DEG C of thermostat water baths and heat 20min, adding enzyme activity is 4000u/g neutral protease 0.3g, temperature 45 C; Be hydrolyzed that to add enzyme activity after 5 hours be 2000u/g food flavor enzyme 0.5g, be adjusted to pH=7.0, remaining same embodiment 1.
4. the extraction of protohemine: with embodiment 1.
Claims (1)
1. utilize yak blood to prepare a method for anhydrous chlorides of rase protoheme, it is characterized in that carrying out according to the enzymolysis of erythrocytic collection, erythrocytic fragmentation, oxyphorase and the extraction step of protohemine; Described in specific as follows:
(1) erythrocytic collection
Collect fresh yak blood 20ml with 50ml test tube, filter with gauze, remove larger impurity, collect filtered liquid, leave standstill after 1 hour after blood coagulation layering, suck the flaxen liquid in upper strata, obtain lower floor red corpuscle coagulum 12ml;
(2) erythrocytic fragmentation
In red corpuscle coagulum, add the deionized water of 0.5 ~ 1.25 times, put into ultrasonic wave and carry out broken 15min, stir with glass stick simultaneously, solid coagulum is fully disperseed, makes erythroclasis simultaneously, release oxyphorase; Solution is poured into fast in six small test tubes, under 60 DEG C of lucifuge conditions, heat 30min, use 3000r/min centrifuge 10min afterwards, the supernatant liquid in six small test tubes is poured in 20ml Erlenmeyer flask, remove the foreign protein of lower floor's sex change, obtain hemoglobin solutions;
(3) enzymolysis of oxyphorase
The HCl0.1mol/L standardized solution of using of hemoglobin solutions is regulated PH=6.8 ~ 7.0, and be put in 50 DEG C of thermostat water baths and heat 20min, adding enzyme activity is 4000u/g neutral protease 0.3g, temperature 45 C; Be hydrolyzed that to add enzyme activity after 5 hours be 2000u/g food flavor enzyme 0.5g, be adjusted to pH=6.8 ~ 7.0, temperature 53 DEG C; After hydrolysis reaction completes, heated and boiled is gone out enzyme;
(4) extraction of protohemine
Oxyphorase is after enzymolysis, peptide chain is disconnected, and has protoheme in hydrolyzed solution, heme peptide and peptide fragment, after adjusting pH=4.5 with HCl0.1mol/L solution, add the sodium acetate solution of mass concentration 1g/100mL, separate out precipitation, collecting precipitation with the centrifugal 10min of 3000r/min rotating speed, use the acetic acid solution of ionized water, volume fraction 50%, the washing of volume fraction 95% ethanolic soln successively once, use washed with diethylether twice again, subzero 20 DEG C of lyophilizes 6 hours, obtain protohemine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094876A (en) * | 2020-09-25 | 2020-12-18 | 中国农业科学院特产研究所 | Preparation method of hemin and composition prepared by same |
| CN115010803A (en) * | 2021-03-03 | 2022-09-06 | 内蒙古天奇生物科技有限公司 | Preparation of hemoglobin polypeptide rich in heme iron |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102659791A (en) * | 2012-04-12 | 2012-09-12 | 武汉普赛特膜技术循环利用有限公司 | Method for extracting hemin and globin from animal blood |
| CN102994584A (en) * | 2012-09-24 | 2013-03-27 | 南昌大学 | Method for producing heme iron by using duck blood |
-
2016
- 2016-03-02 CN CN201610117183.4A patent/CN105567759A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102659791A (en) * | 2012-04-12 | 2012-09-12 | 武汉普赛特膜技术循环利用有限公司 | Method for extracting hemin and globin from animal blood |
| CN102994584A (en) * | 2012-09-24 | 2013-03-27 | 南昌大学 | Method for producing heme iron by using duck blood |
Non-Patent Citations (2)
| Title |
|---|
| 李晨光等: ""动物血液血红素铁提取方法研究"", 《食品工业科技》 * |
| 贾志春等: ""木瓜蛋白酶酶解牦牛血红蛋白制备氯化血红素关键工艺研究"", 《食品工业科技》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112094876A (en) * | 2020-09-25 | 2020-12-18 | 中国农业科学院特产研究所 | Preparation method of hemin and composition prepared by same |
| CN115010803A (en) * | 2021-03-03 | 2022-09-06 | 内蒙古天奇生物科技有限公司 | Preparation of hemoglobin polypeptide rich in heme iron |
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