CN102659791B - Method for extracting hemin and globin from animal blood - Google Patents
Method for extracting hemin and globin from animal blood Download PDFInfo
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- CN102659791B CN102659791B CN201210106042.4A CN201210106042A CN102659791B CN 102659791 B CN102659791 B CN 102659791B CN 201210106042 A CN201210106042 A CN 201210106042A CN 102659791 B CN102659791 B CN 102659791B
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Abstract
The invention relates to a method for extracting hemin and globin by the use of leech protease with animal blood as a raw material. The method comprises the following main steps of: 1, carrying out middle-temperature extraction to prepare leech protease for later use; 2, taking fresh animal blood, carrying out anticoagulation treatment, centrifuging and collecting hemoglobin cells; 3, adding one time of pure water, carrying out ultrasonic treatment and breaking cells; 4, filtering through a screen mesh, leaving a filtrate, and adjusting pH value; 5, adding leech protease for enzymatic hydrolysis; 6, adjusting pH value of an enzymatic hydrolysis liquid and carrying out centrifugal separation; and 7, washing a sediment which has undergone centrifugation, carrying out dehydrolysis and drying to obtain hemin; letting a supernatant which has undergone centrifugation pass through a microfiltration membrane to remove impurities, using a nanofiltration membrane for concentration, and carrying out dehydrolysis and drying on a concentrate to obtain globin. According to the invention, animal blood resources are fully utilized and the leech extract product is used as an enzyme preparation to produce hemin and globin with high added values. The technology is simple and easy to operate, the production cost is low, and the method is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of production method of livestock blood being carried out to intensive processing, relating generally to a kind of is raw material with livestock blood, utilizes leech proteolytic enzyme to extract the method for protohemine and globin peptide.
Background technology
Livestock and poultry blood content is different because of animal species, for pig blood, its weight be about live pig live body heavy 3% ~ 5%.The blood can collected after slaughtered animals accounts for total amount 60% ~ 70%, and remaining is stranded in liver, kidney, skin and body.Usually butcher a pig, about can collect 2.0 ~ 3.0kg blood.
China recognizes that from the seventies livestock blood has better nutritivity health value gradually, but limits by factors, is still far from perfect to its utilization, causes a large amount of valuable livestock and poultry blood wasting of resources, and causes environmental pollution.The latter stage seventies and the initial stage eighties, country starts the comprehensive utilization paying attention to livestock blood, it can be used as emphasis problem to tackle key problems, and development research goes out some livestock blood goods in succession.Obtained remarkable progress in recent years, in succession there is many great scientific payoffss, its economic benefit and social benefit are increased substantially, especially the Study and appliance in the projects such as animal and fowl fodder, nutritional supplement, protoheme, hematoporphyrin derivative, amino-acid nutrition liquid, reaches advanced world standards.
In past 10 years, the regionalization trend of Chinese livestock rearing is day by day obvious, and each department are all concentrated in implementation and butchered system in addition, and livestock blood source is concentrated, extensive and safe and reliable, therefore makes full use of livestock blood and seems particularly important in China.How this precious resources of rational exploitation and utilization, cause the great attention of domestic relevant department, especially the focus of the protoheme that all plays an important role in multiple industry numerous research is especially extracted in, utilize livestock blood to prepare high purity protohemine and globin peptide and have very important status for raising livestock blood comprehensive utilization degree, also be existing economic worth simultaneously, have again the cause of social value.
Protoheme is the reactive site of oxyphorase, is the important natural porphyrin iron cpd of a class, is extensively present in the blood of higher animal, muscle.The heme products extracted from animal blood is generally that protohemin is called for short protohemine, i.e. the protohemine that formed in conjunction with a chlorion of protoporphyrin.Protohemine is used for the treatment of hypoferric anemia, not only evident in efficacy, and curative ratio is high and without gastrointestinal side effect, and easy administration is easy to patients, is the best iron supplementary of current curative effect.
Globin peptide is that oxyphorase removes the little peptide of the protein part after protoheme through enzymolysis, more easily, sooner absorbed by body than total free aminoacids completely, in Promote immunity hyperplasia, antitumor, anti-oxidant, enhancing body immunizing power etc., have certain effect.
Extraction protohemine is common and the method for technical maturity mainly contains sodium-acetate method, distillation method, tannic acid method, carboxymethyl cellulose method and ice acetic acid method.But in principle, sodium-acetate method, distillation method, tannic acid method are all utilize acetone to be prepared.Its technical process is complicated, difficult solvent recovery, is not too applicable to large-scale industrial and produces, and understands the residual of organic solvent in product, there is certain potential safety hazard.
Adopting the method for biological enzymolysis the protoheme in livestock blood and protein to be separated, is a kind of comparatively feasible technical scheme.But higher owing to market being bought suitable protease preparation price, on industrial production cost impact greatly.
Summary of the invention
In order to overcome problem and the defect of existing technique, making full use of livestock blood resource, the invention provides a kind of protohemine and globin peptide are extracted in utilization from livestock blood method using Hirudo extract as protease preparation.
Leech, popular name leech, growth and breeding in the freshwater of inland is the traditional medicinal hydrocoles of special type of China, its dry products concoct after the traditional Chinese medical science be used as medicine, there is the effect such as treatment apoplexy, hypertension, the clear stasis of blood, amenorrhoea, wound.Record in ancient medical book and utilize leech to treat various diseases, call its " main by extravesated blood, hemostasis, close by the moon, broken blood disappears and gathers ", cure its eliminating pathogenic factor for supporting vital QI of holy Zhang Zhongjing, the disease for the treatment of " hemostasis ", " water knot ", shows the curative effect of its uniqueness.Adult leech mainly with the blood of animal for food, the proteolytic enzyme contained by its Digestive tract can efficiently enzymolysis eat protein in blood, be beneficial to digest and assimilate.
The present invention mainly utilizes and the protoheme livestock blood and protein is separated as protease preparation from Hirudo extract, and comprehensive ultrasonic technology and the membrane separation technique of adopting obtains high purity protohemine and globin peptide totally two kinds of products.
Can being realized by following technical proposals of day of the present invention:
Utilize leech proteolytic enzyme from livestock blood, extract the method for protohemine and globin peptide, it is characterized in that carrying out according to the following steps:
A, leech to be cleaned, dry, chopping;
B, by a step chopping after leech put into the sodium citrate buffer solution that concentration is 0.05% ~ 0.12%, solid-liquid ratio is 1: 5, and pH value is 5.0 ~ 6.5, and temperature is 35 ~ 50 DEG C, soaks 10 ~ 30 minutes, centrifugation;
Adding ammonium sulfate degree of reaching capacity in c, the supernatant liquor that centrifugation in b step obtained is 30% ~ 50%, and carry out fractionation precipitation, the supernatant liquor obtained is that leech protein enzyme solution is for subsequent use;
D, by fresh livestock blood in 0.8% ~ 1.0% ratio add trisodium citrate and make antithrombotics, Homogeneous phase mixing 10 minutes, then centrifugation, abandoning supernatant, collect lower floor's oxyphorase cell;
E, by the oxyphorase cell collected in Step d according to 1: 1 ratio mix with pure water, ultrasonication 15 ~ 20 minutes smudge cellses;
F, the cytoclasis liquid in step e to be filtered through 100 eye mesh screens, leave filtrate adjust ph to 2.0 ~ 3.5;
G, by mix up pH value in f step cytoclasis liquid in rapid temperature increases to 85 ~ 95 DEG C, be incubated 10 ~ 15 minutes, make the abundant sex change of protein; The leech protein enzyme solution that ratio in 5% adds by obtaining in step c carries out enzymolysis, and adjust ph is 5.0 ~ 6.5, and enzymolysis time is 100 ~ 180 minutes, keeps temperature to be 40 ~ 60 DEG C;
H, the enzymolysis solution that g step obtains is warmed up to 85 ~ 90 DEG C of enzymes that go out, the time is about 5 minutes, adjust ph to 4.0 ~ 5.5, and the throw out that centrifugation obtains dehydrates again and namely obtains protohemine after washing;
I, supernatant liquor centrifugation in h step obtained filter through microfiltration membrane device, impurity trapped, fiber and high molecular weight protein etc., and obtain microfiltration membrane filtrate, micro-filtrate membrane filtration aperture is 0.1 ~ 1.0um;
J, the microfiltration membrane filtrate obtained in i step concentrated through nano filter membrance device, for the follow-up operation that dehydrates reduces the heavy burdens, nanofiltration membrane aperture is 0.1 ~ 1.0nm again;
K, again the nanofiltration membrane concentrated solution obtained in j step to be dehydrated, obtain globin peptide.
Sodium citrate buffer solution concentration in described step b is 0.08% ~ 0.10%, and pH value is 5.5 ~ 6.0, and temperature is 40 ~ 45 DEG C, soaks 15 ~ 20 minutes.
Cytoclasis liquid filtrate adjust ph in described step f is 2.5 ~ 3.0.
Enzymolysis process adjust ph in described step g is 5.5 ~ 6.0, and enzymolysis time is 120 ~ 150 minutes, keeps temperature to be 45 ~ 50 DEG C.
The enzymolysis solution pH value after enzyme of going out in described step h is adjusted to 4.5 ~ 5.0.
The present invention, compared with existing technique, has the following advantages:
The present invention effectively make use of livestock blood resource, adopts biological enzyme to combine with ultrasonic technology, membrane separation technique, extracts protohemine and globin peptide.
The present invention utilizes Hirudo extract carried out degrading by the protein in livestock blood as protease preparation and split with protoheme, reduces the production cost of enzymolysis and extraction technique.
Present invention process is simple, operational safety, can obtain the target product of high-quality.
The outward appearance of the protohemine that the present invention obtains is black powder, and purity is greater than 90%; The outward appearance of globin peptide is pink, and lipidated protein is greater than 90%, and its molecular weight is mainly distributed between 200 ~ 1000Dal.
Embodiment
Below in conjunction with embodiment, the present invention will be described in detail:
Embodiment 1:
Water intaking leech 100g, dries after cleaning; Chopping; Put into the sodium citrate buffer solution that concentration is 0.08%, solid-liquid ratio is 1: 5, and pH value is 5.5, and temperature is 40 DEG C, soaks 15 minutes; Centrifugation, collects supernatant liquor; Supernatant liquor being added ammonium sulfate degree of reaching capacity is 30%, carries out fractionation precipitation, and the supernatant liquor leech protein enzyme solution obtained is for subsequent use.Get 200ml fresh pig blood, the ratio according to 0.8% adds antithrombotics trisodium citrate, and Homogeneous phase mixing centrifugation after 10 minutes, removes supernatant liquor, collects lower floor's oxyphorase cell, about about 100ml; Pure water is added again, ultrasonication 15 minutes smudge cellses according to the ratio of 1: 1; Broken liquid is filtered through 100 eye mesh screens, leaves filtrate, slowly regulate filtrate pH value to 2.5 with dilute hydrochloric acid; Rapid temperature increases to 85 DEG C, is incubated 10 minutes, makes the abundant sex change of protein; Ratio in 5% adds the leech protein enzyme solution prepared and carries out enzymolysis, adjust ph to 5.5, and enzymolysis time is 120 minutes, keeps temperature to be 45 DEG C; Be warmed up to 85 DEG C of enzymes that go out after enzymolysis completes again, the time is about 5 minutes; Regulate enzymolysis solution pH value to 4.5, after centrifugation, be precipitated thing and supernatant liquor respectively.Throw out dehydrates and can obtain protohemine after washing; And the microfiltration membrane that the first aperture after filtration of supernatant liquor is 0.2um carries out removal of impurities, obtain concentrated solution and filtrate respectively; Filtrate concentrates by the nanofiltration membrane that pore size filter is 0.2nm again; Finally concentrated solution is dehydrated, globin peptide can be obtained.
Embodiment 2:
Water intaking leech 2kg, dries after cleaning; Chopping; Put into the sodium citrate buffer solution that concentration is 0.10%, solid-liquid ratio is 1: 5, and pH value is 6.0, and temperature is 45 DEG C, soaks 20 minutes; Centrifugation, collects supernatant liquor; Supernatant liquor being added ammonium sulfate degree of reaching capacity is 50%, carries out fractionation precipitation, and the supernatant liquor leech protein enzyme solution obtained is for subsequent use.Get the new freshly-slaughtered poultry blood of 100kg, the ratio according to 1.0% adds antithrombotics trisodium citrate, and Homogeneous phase mixing centrifugation after 10 minutes, removes supernatant liquor, collects lower floor's oxyphorase cell, about about 50kg; Pure water is added again, ultrasonication 20 minutes smudge cellses according to the ratio of 1: 1; Broken liquid is filtered through 100 eye mesh screens, leaves filtrate, slowly regulate filtrate pH value to 3.0 with dilute hydrochloric acid; Rapid temperature increases to 95 DEG C, is incubated 15 minutes, makes the abundant sex change of protein; Ratio in 5% adds the leech protein enzyme solution prepared and carries out enzymolysis, adjust ph to 6.0, and enzymolysis time is 150 minutes, keeps temperature to be 50 DEG C; Be warmed up to 90 DEG C of enzymes that go out after enzymolysis completes again, the time is about 5 minutes; Regulate enzymolysis solution pH value to 5.0, after centrifugation, be precipitated thing and supernatant liquor respectively.Throw out dehydrates and can obtain protohemine after washing; And the microfiltration membrane that the first aperture after filtration of supernatant liquor is 0.5um carries out removal of impurities, obtain concentrated solution and filtrate respectively; Filtrate concentrates by the nanofiltration membrane that pore size filter is 0.5nm again; Finally concentrated solution is dehydrated, globin peptide can be obtained.
Claims (1)
1. from livestock blood, extract a method for protohemine and globin peptide, it is characterized in that carrying out according to the following steps:
A, leech to be cleaned, dry, chopping;
B, by a step chopping after leech put into the sodium citrate buffer solution that volumetric concentration is 0.08% ~ 0.10%, solid-liquid ratio is 1:5, and pH value is 5.5 ~ 6.0, and temperature is 40 ~ 45 DEG C, soaks 15 ~ 20 minutes, centrifugation;
Adding ammonium sulfate degree of reaching capacity in c, the supernatant liquor that centrifugation in b step obtained is 30% ~ 50%, and carry out fractionation precipitation, the supernatant liquor obtained is that leech protein enzyme solution is for subsequent use;
D, by fresh livestock blood by volume the ratio of concentration 0.8% ~ 1.0% add trisodium citrate and make antithrombotics, Homogeneous phase mixing 10 minutes, then centrifugation, abandoning supernatant, collect lower floor's oxyphorase cell;
E, the oxyphorase cell collected in Step d to be mixed with pure water according to the ratio of 1:1, ultrasonication 15 ~ 20 minutes smudge cellses;
F, the cytoclasis liquid in step e to be filtered through 100 eye mesh screens, leave filtrate adjust ph to 2.5 ~ 3.0;
G, by mix up pH value in f step cytoclasis liquid in rapid temperature increases to 85 ~ 95 DEG C, be incubated 10 ~ 15 minutes, make the abundant sex change of protein; Be that the leech protein enzyme solution that the ratio of 5 ﹪ adds by obtaining in step c carries out enzymolysis by volume, adjust ph is 5.5 ~ 6.0, and enzymolysis time is 120 ~ 150 minutes, keeps temperature to be 45 ~ 50 DEG C;
H, the enzymolysis solution that g step obtains is warmed up to 85 ~ 90 DEG C of enzymes that go out, the time is 5 minutes, adjust ph to 4.5 ~ 5.0, and the throw out that centrifugation obtains dehydrates again and namely obtains protohemine after washing, and purity is greater than 90%;
I, supernatant liquor centrifugation in h step obtained filter through microfiltration membrane device, and impurity trapped, fiber and high molecular weight protein, obtain microfiltration membrane filtrate, and micro-filtrate membrane filtration aperture is 0.1 ~ 1.0um;
J, the microfiltration membrane filtrate obtained in i step concentrated through nano filter membrance device, for the follow-up operation that dehydrates reduces the heavy burdens, nanofiltration membrane aperture is 0.1 ~ 1.0nm again;
K, dehydrated by the nanofiltration membrane concentrated solution obtained in j step, obtain globin peptide, lipidated protein is greater than 90% again.
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| US9809555B2 (en) * | 2015-12-02 | 2017-11-07 | Rotam Agrochem International Company Limited | Form of mefenpyr-diethyl, a process for its preparation and use of the same |
| CN106929554A (en) * | 2015-12-31 | 2017-07-07 | 上海杰隆生物制品股份有限公司 | A kind of preparation method of globin peptide |
| CN105567759A (en) * | 2016-03-02 | 2016-05-11 | 兰州天和生物催化技术有限公司 | Method for preparing anhydrous chlorhematin by utilizing yak blood |
| CN105768110A (en) * | 2016-03-31 | 2016-07-20 | 常州大学 | Methodof extracting human body trace element from waste blood |
| CN108998492B (en) * | 2018-08-31 | 2022-07-19 | 南京钦润生物科技有限公司 | Preparation method of globin |
| 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 |
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| JPS59181279A (en) * | 1983-03-29 | 1984-10-15 | Sato Yakugaku Kenkyusho:Kk | Preparation of hemin |
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| CN101332211A (en) * | 2008-08-06 | 2008-12-31 | 山东大学 | Leech extract and preparation method and use thereof |
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Inventor after: Wan Duanji Inventor after: Zou Huarong Inventor after: Wu Yao Inventor after: Yang Tao Inventor after: Ma Chao Inventor after: Xu Lipeng Inventor after: Liao Yujie Inventor after: Zhu Lin Inventor after: Wu Zhengqi Inventor after: Xu Guonian Inventor after: Li You Inventor after: Xiao Shiying Inventor after: Wang Xiong Inventor after: Si Jia Inventor after: Cai Jun Inventor before: Wan Duanji Inventor before: Wu Yao Inventor before: Yang Tao Inventor before: Ma Chao Inventor before: Xu Lipeng Inventor before: Liao Yujie Inventor before: Wu Zhengqi Inventor before: Xu Guonian Inventor before: Li You Inventor before: Xiao Shiying Inventor before: Wang Xiong Inventor before: Si Jia Inventor before: Cai Jun Inventor before: Zou Huarong |
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