CN110229862A - A kind of Yak-skin Gelatin preparation method promoting platelet activation - Google Patents
A kind of Yak-skin Gelatin preparation method promoting platelet activation Download PDFInfo
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- 239000008273 gelatin Substances 0.000 title claims abstract description 50
- 230000010118 platelet activation Effects 0.000 title claims abstract description 26
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 230000001737 promoting effect Effects 0.000 title claims abstract description 20
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The present invention relates to a kind of Yak-skin Gelatin preparation methods for promoting platelet activation, comprising the following steps: (1) cleans up the yak in Qinghai-tibet skin of fresh band hair, the yak skin through ultraviolet light irradiation, after being irradiated；(2) the yak skin after irradiating is cut into the block of 1 ~ 3cm, takes out after extracting in boiling water, pierces through yak skin with thorniness plate；(3) by step, (2) resulting yak skin is put into mass concentration to digest in 0.5% ~ 8% enzymolysis liquid, filters, obtains filtrate；(4) active carbon is added in filtrate, boils, filter after being sufficiently stirred, respectively obtain enzymatic hydrolysis solution and filter residue for the first time；(5) filter residue is put into the enzymolysis liquid that mass concentration is 1% ~ 10% and digests, and obtains second of enzymatic hydrolysis solution；(6) boiled 5 ~ 40 minutes after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution, be centrifuged, obtain supernatant；Supernatant use hollow-fibre membrane ultrafiltration, obtain Yak-skin Gelatin original protein solution, the Yak-skin Gelatin original protein solution it is freeze-dried to get.The method of the present invention is simple, easy to implement.
Description
Technical field
The present invention relates to a kind of preparation process of collagen more particularly to a kind of Yak-skin Gelatins for promoting platelet activation
Preparation method.
Background technique
Currently, being always to carry out with the collagen after losing hair or feathers for the research of yak skin, the effect of yak hair is had ignored.
Ox hair is mainly made of keratin, has amino acid long-chain, and cyokeratin is similar to collagen, and there are two types of major protein, Yi Zhongwei
Containing the more cytoplasm albumen of element sulphur, contain cystine；Another kind is the less cellulosic albumen of sulfur-bearing.
Cystine is a kind of sulfur-containing amino acid, mostly contained in keratin, is one of amino acid needed by human, does not have to human body
There is ill effect.Pharmaceutically have and body cell is promoted to aoxidize and restore function, increase white blood cell and prevents the works such as pathogen development
With being also used for the acute infectious diseases such as dysentery, typhoid fever, influenza, asthma, neuralgia, eczema and various poisoning illness etc., and have dimension
Hold the effect of protein configuration.
Platelet aggregation is related to various adhesion molecules, including II b/ of Gp, III a, the von Willebrand factor, P-
Selection etc. (Massberg etc., 1998；Frenette etc., 2000).Wherein, the Weibel-Palade body of endothelial cell and
There are P-selection(Skinner etc. in the granule of platelet, 1990；Frenette etc., 1995；Romo etc., 1999).In blood
In platelet accumulation process P-selection first rapidly mobilize arrive cell surface, then with P-selection protein ligands -1
(pgp -1) is integrated on the blood platelet of activation (Larsen etc., 1989).Due to P-selection sensibility with higher and
Specificity, it has also become one of the index of generally acknowledged detection platelet activation.
I a/ of Gp, II a is collagen receptor one of of the platelet surface in conjunction with collagen, and the first step of hemostasis is exactly
Started by I a/ of Gp, II a, internal/external signal is caused to be transduceed, activation collagen receptor, so that activation blood platelet (Nieswandt etc., 2001；
Arthur etc., 2007).
Summary of the invention
A kind of technical problem to be solved by the invention is to provide methods promotion platelet activation simple, easy to implement
Yak-skin Gelatin preparation method.
To solve the above problems, a kind of Yak-skin Gelatin preparation method for promoting platelet activation of the present invention, including
Following steps:
(1) the fresh yak in Qinghai-tibet skin for taking away 1 ~ 2 centimetre of hair of skin is cleaned up, through ultraviolet light irradiation, after being irradiated
Yak skin；
(2) the yak skin after the irradiation is cut into the block of 1 ~ 3cm, takes out after extracting 5 ~ 30 minutes in boiling water, is pierced through with thorniness plate
Yak skin；
(3) by the step, (2) resulting yak skin is put into mass concentration to digest in 0.5% ~ 8% enzymolysis liquid, filters, is filtered
Liquid；The mass ratio of the yak skin and the enzymolysis liquid is 1:5 ~ 1:20；
(4) the active carbon of its quality 3 ~ 10% is added in the filtrate, is boiled after being sufficiently stirred 5 ~ 40 minutes, filters, respectively obtains
Enzymatic hydrolysis solution and filter residue for the first time；
(5) the filter residue is put into the enzymolysis liquid that mass concentration is 1% ~ 10% and digests, and obtains second of enzymatic hydrolysis solution；The filter residue
Mass ratio with the enzymolysis liquid is 1:8 ~ 1:20；
(6) boil 5 ~ 40 minutes, be centrifuged after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution,
Obtain supernatant；
(7) the supernatant uses hollow-fibre membrane ultrafiltration, obtains Yak-skin Gelatin original protein solution, the Yak-skin Gelatin original albumen is molten
Liquid it is freeze-dried to get.
(1) middle ultraviolet lamp irradiation condition refers to that radiation wavelength is 200nm ~ 275nm to the step, and irradiation time is 0.3 ~ 2.0
Hour.
The step enzymolysis liquid that (3) middle mass concentration is 0.5% ~ 8% refers to addition 5 ~ 80g albumen in 1L deionized water
Enzyme, solution obtained by being uniformly mixed.
(3) middle enzymatic hydrolysis condition refers to that temperature is 30 ~ 60 DEG C to the step, and the time is 0.5 ~ 36 hour.
The step enzymolysis liquid that (5) middle mass concentration is 1% ~ 10% refers to addition 10 ~ 100g albumen in 1L deionized water
Enzyme, solution obtained by being uniformly mixed.
(5) middle enzymatic hydrolysis condition refers to that temperature is 30 ~ 55 DEG C to the step, and the time is 0.5 ~ 24 hour.
The protease refers to thiol protease, bromelain, streptomycete Keratinase, saccharomycete enzyme, stomach cardia
Enzyme, trypsase, cathepsin, papain, subtilopeptidase A, alkali protease, one in compound protease
Kind or two kinds or more of compound.
(6) the middle condition being centrifugated refers to that rate is 15000 ~ 25000rpm to the step, and the time is 5 ~ 60min.
The step (7) in hollow-fibre membrane aperture be 3.5 ~ 7.0 μm.
(7) the middle condition being freeze-dried refers to that temperature is -10 ~ -50 DEG C to the step, and the time is 2 ~ 48 hours.
Compared with the prior art, the present invention has the following advantages:
1, the Yak-skin Gelatin original albumen in the present invention is using peculiar skin of the ox kind yak with hair in Qinghai-Tibet Platean as raw material, after separation
Obtained molecular weight is the pure natural collagen of 10 ~ 20kDa.The exploitation of Yak-skin Gelatin original albumen makes the spy of Qinghai-Tibet Platean
There is resource yak more preferably to be utilized.
2, the Yak-skin Gelatin original albumen prepared by the present invention is proved through pharmacodynamic experiment, can be by promoting in the granule of platelet
Tolerant release accelerates platelet adhesion reaction-allosteric-release-aggregation process to complete platelet activation, promotees in early stage activation blood platelet
Into platelet adhesion reaction, increase the expression quantity of serum Gp I a/II a, Gp II a of b/III and Gp IV, P-selection, CD62P activation
Rate reaches 22.58%.
1. influence of the Yak-skin Gelatin to mouse P-selection:
Yak-skin Gelatin can intervene hemoglutination, and blood platelet participates in hemostasis and coagulation process, and blood platelet is in the position of impaired vascular wall
Set adherency and activation.Platelet activation is hemostasis since angiorrhoxis, fibrin, fibrin ferment participation under,
A series of physiological reactions (Ye etc., 2004) such as conversion, dry, release, aggregation occur rapidly after being stimulated for blood platelet.
60 KM mouse are randomly divided into 6 groups, blank control group, Yak-skin Gelatin A1, A2, A3, A4 administration group (2 g/kg/
D), Yunnan Baiyao Capsule administration group (1405.62 mg/kg/d), successive administration 7 days.It extracts eyeball method and takes blood, 3000 r/min,
4 DEG C of 20 min of centrifugation, after drawing supernatant, according to P- in mouse P-selection enzyme-linked immunosorbent assay mice serum
The content of selection.
It is compared as shown in Figure 1 with control group, it is aobvious that different molecular weight Yak-skin Gelatin administration group P-selection increases expression
It writes raising (P<0.01) and compares no difference (P>0.05) with Yunnan Baiyao Capsule administration group.Prompt the administration of each group Yak-skin Gelatin
Afterwards, P-selection mobilizes number to increase on platelet membrane, and the expression of P-selection can represent σ, blood platelet on platelet membrane
The burst size (such as Cauwenberghs, 2000) of grain, these blood platelets participate in rapidly essential thrombocythemia aggregation, then and are permitted
Multiple ligand combines, and promotes cascade reaction.This result is consistent with the result of bleeding and reduced clotting time, and P-selection exists
The lower Yak-skin Gelatin group expression of molecular weight distribution is higher, illustrates that the active component of Yak-skin Gelatin is that molecular weight distribution is lower
Section.
2. influence of the Yak-skin Gelatin to I a/ of rat platelet Gp, II II b/ of a, Gp, III a and Gp IV:
40 SD rats are randomly divided into 4 groups, blank control group, Yak-skin Gelatin 1,2 administration groups, Yunnan Baiyao Capsule administration group,
Successive administration 7 days, after 30 min of last dose, using 10% chloral hydrate anesthesia rat, heart extracting blood was anticoagulant, 1000 r centrifugation
10 min prepare Platelet-rich plasm, measure rat according to I a/ of rat Gp, II II b/ of a, Gp, III a and Gp IV enzyme linked immunological book
The content of I a/ of Gp, II II b/ of a, Gp, III a and Gp IV in serum.
The expression of platelet membrane proteins and the activation of blood platelet are related, especially Gp I a/II a, Gp II a of b/III and Gp IV
Whether all the release of early stage particle is with platelet activation directly in reaction hemostasis.
When subendothelial collagen promotes platelet adhesion reaction and activation, blood platelet is captured, I a/ of platelet membrane proteins, II a,
II b/ of Gp, III a and IV participates (Fuster etc., 1992).Table 1 with control group the result shows that compare, Yak-skin Gelatin administration group
I a/ of Gp, II a expression raising (P< 0.01), and consistent with Yunnan Baiyao Capsule group trend.
II b/ of Gp, III a passes through the von Willebrand factor indirectly in conjunction with collagen receptor, can be anti-with vitamin A acid, blood platelet
Albumen, fibrin and fibronectin is answered to combine (Peter etc., 2004).II b/ of Gp, III a great expression on blood platelet, each
Blood platelet has 5 ~ 80,000, accounts for about 1% ~ 2%(Phillips etc. of blood platelet albumen total amount, and 1988；Wagner etc., 1996).GpⅡ
III a of b/ mainly generates ecto-entad signal when there are conditions such as apparent clot retracts and coagulant, so that II b/ of Gp, III a believes
Number with height sensibility and specificity (Pello etc., 2012).The display of table 1 is compared with control group, and Yak-skin Gelatin can increase
II b/ of Gp, III a expression (P<0.05)。
Gp IV is widely present in various cells and tissue, such as blood platelet, vascular endothelial cell and smooth muscle cell etc..
There are about 12000 ~ 15000 Gp IV, molecular weight is 88 kD on platelet membrane surface.Gp IV is by acting on fyn, lyn and yes
Protein tyrosine kinase participates in the interaction of platelet aggregation and blood platelet and monokaryon induced platelet signal transduction
(Sarratt etc., 2005；Mo., 2012).The occurred conformation in early stage platelet adhesion reaction of Gp IV changes, exposure binding site, association
Help blood platelet incorporating collagen albumen.Table 1 display compared with control group, Yak-skin Gelatin can increase Gp IV expression (P<0. 05)。
The influence of rat platelet GpIa/IIa, GpIIb/IIIa and Gp IV after the administration of 1 yak skin of table
Note: the expression difference containing same letter is not significant (P > 0.05), expression significant difference containing different letters (P <
0.05).
3. influence of the Yak-skin Gelatin to rat platelet CD62P:
Carry out rapid evaluation (Ramstr m with the receptors such as Flow cytometry CD62P and platelet-monocyte compound
Equal, 2016).CD62P is stored in the Ca in Platelet alpha granule and Weibel-Palade endothelial cell2+Rely on receptor
(Furie etc., 2001；McEver etc., 2002).
When coagulation process starts, the glycoprotein in α particle is promptly released into blood plasma, and CD62P is in platelet surface table
It reaches, the level of the antibody test CD62P of fluorescent marker at this moment can be used, this is the most special, most sensitive of platelet activation
Index (Hamburger etc., 1990；Leytin etc., 2000；Conway etc., 2003；Lee etc., 2008；Wachowicz etc.,
2002；Kutlar etc., 2014).
Shown in table 2, the expression of Yak-skin Gelatin CD62P be apparently higher than control group (P< 0.01)。
2 Yak-skin Gelatin of table to rat platelet CD62P influence (n = 8, )
Note: compared to the blank group* P< 0.05,** P<0.01。
3, the method for the present invention is simple, easy to implement.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
Fig. 1 be Yak-skin Gelatin of the present invention to mouse P-selection influence (n =10,), * compared to the blank group
P < 0.01 P < 0.05, * *.
Specific embodiment
A kind of Yak-skin Gelatin preparation method for promoting platelet activation of embodiment 1, comprising the following steps:
(1) the fresh yak in Qinghai-tibet skin for taking away 2 centimetres of hairs of skin is cleaned up, shone with the radiation wavelength of 275nm through ultraviolet lamp
It penetrates 2.0 hours, the yak skin after being irradiated.
(2) the yak skin after irradiating is cut into the block of 3cm, takes out after extracting 30 minutes in boiling water, pierces through yak with thorniness plate
Skin.
(3) by step, (2) resulting yak skin is put into the enzymolysis liquid that mass concentration is 0.5%, is digested 8 hours in 45 DEG C, mistake
Filter, obtains filtrate.
Wherein: the enzymolysis liquid that mass concentration is 0.5% refers in 1L deionized water addition 5g protease, be uniformly mixed and
The solution obtained.Protease refers to 2.5g thiol protease, 0.25g alkali protease, 1.5g compound protease, 0.5g streptomycete angle
The mixture of matter protease, 0.25g saccharomycete enzyme.
The mass ratio (g/g) of yak skin and enzymolysis liquid is 1:5.
(4) the active carbon of its quality 3% is added in filtrate, is boiled after being sufficiently stirred 40 minutes, filters, respectively obtains for the first time
Digest solution and filter residue.
(5) filter residue is put into the enzymolysis liquid that mass concentration is 10%, digests 24 hours in 55 DEG C, it is molten to obtain second of enzymatic hydrolysis
Liquid.
Wherein: the enzymolysis liquid that mass concentration is 10% refers in 1L deionized water addition 100g protease, be uniformly mixed and
The solution obtained.Protease refers to 30g bromelain, 10g pepsin, 5g trypsase, 20g cathepsin, 10g pawpaw
The mixture of protease, 25g subtilopeptidase A.
The mass ratio (g/g) of filter residue and enzymolysis liquid is 1:8.
(6) enzymatic hydrolysis solution will digest after solution merges and boil 40 minutes with second for the first time, with the rate of 15000rpm from
The heart separates 5min, obtains supernatant.
(7) supernatant uses aperture for 7.0 μm of hollow-fibre membrane ultrafiltration, obtains Yak-skin Gelatin original protein solution, the yak
Collagen solution in -10 DEG C be freeze-dried 2 hours to get.
A kind of Yak-skin Gelatin preparation method for promoting platelet activation of embodiment 2, comprising the following steps:
(1) the fresh yak in Qinghai-tibet skin for taking away 1 centimetre of hair of skin is cleaned up, shone with the radiation wavelength of 200nm through ultraviolet lamp
It penetrates 0.3 hour, the yak skin after being irradiated.
(2) the yak skin after irradiating is cut into the block of 1cm, extracts in boiling water and takes out after five minutes, pierces through yak with thorniness plate
Skin.
(3) by step, (2) resulting yak skin is put into the enzymolysis liquid that mass concentration is 8%, is digested 0.5 hour in 30 DEG C, mistake
Filter, obtains filtrate.
Wherein: the enzymolysis liquid that mass concentration is 8% refers to the addition 80g protease in 1L deionized water, is uniformly mixed and obtains
Solution.Protease refers to 2g bromelain, 30g pepsin, 10g trypsase, 8g cathepsin, 16g pawpaw egg
The mixture of white enzyme, 14g subtilopeptidase A.
The mass ratio (g/g) of yak skin and enzymolysis liquid is 1:20.
(4) the active carbon of its quality 10% is added in filtrate, is boiled after being sufficiently stirred 5 minutes, filters, respectively obtains for the first time
Digest solution and filter residue.
(5) filter residue is put into the enzymolysis liquid that mass concentration is 1%, digests 0.5 hour in 30 DEG C, it is molten to obtain second of enzymatic hydrolysis
Liquid.
Wherein: the enzymolysis liquid that mass concentration is 1% refers to the addition 10g protease in 1L deionized water, is uniformly mixed and obtains
Solution.Protease refers to 2g thiol protease, 0.5g alkali protease, 1.5g compound protease, 3g streptomycete keratoprotein
The mixture of enzyme, 3g saccharomycete enzyme.
The mass ratio (g/g) of filter residue and enzymolysis liquid is 1:20.
(6) enzymatic hydrolysis solution will digest after solution merges and boil 5 minutes with second for the first time, with the rate of 25000rpm from
The heart separates 60min, obtains supernatant.
(7) supernatant uses aperture for 3.5 μm of hollow-fibre membrane ultrafiltration, obtains Yak-skin Gelatin original protein solution, the yak
Collagen solution in -50 DEG C be freeze-dried 48 hours to get.
A kind of Yak-skin Gelatin preparation method for promoting platelet activation of embodiment 3, comprising the following steps:
(1) the fresh yak in Qinghai-tibet skin for taking away 1.5 centimetres of hairs of skin is cleaned up, with the radiation wavelength of 223nm through ultraviolet lamp
Irradiation 1.0 hours, the yak skin after being irradiated.
(2) the yak skin after irradiating is cut into the block of 2cm, takes out after extracting 15 minutes in boiling water, pierces through yak with thorniness plate
Skin.
(3) by step, (2) resulting yak skin is put into the enzymolysis liquid that mass concentration is 5%, is digested 4 hours in 40 DEG C, mistake
Filter, obtains filtrate.
Wherein: the enzymolysis liquid that mass concentration is 5% refers to the addition 50g protease in 1L deionized water, is uniformly mixed and obtains
Solution.Protease refers to the mixture of 20g subtilopeptidase A, 10g thiol protease, 20g alkali protease.
The mass ratio (g/g) of yak skin and enzymolysis liquid is 1:15.
(4) the active carbon of its quality 5% is added in filtrate, is boiled after being sufficiently stirred 20 minutes, filters, respectively obtains for the first time
Digest solution and filter residue.
(5) filter residue is put into the enzymolysis liquid that mass concentration is 6%, is digested 8 hours in 50 DEG C, is obtained second of enzymatic hydrolysis solution.
Wherein: the enzymolysis liquid that mass concentration is 6% refers to the addition 60g compound protease in 1L deionized water, is uniformly mixed
Obtained by solution.
The mass ratio (g/g) of filter residue and enzymolysis liquid is 1:10.
(6) enzymatic hydrolysis solution will digest after solution merges and boil 30 minutes with second for the first time, with the rate of 15000rpm from
The heart separates 30min, obtains supernatant.
(7) supernatant uses aperture for 5.0 μm of hollow-fibre membrane ultrafiltration, obtains Yak-skin Gelatin original protein solution, the yak
Collagen solution in -10 DEG C be freeze-dried 48 hours to get.
A kind of Yak-skin Gelatin preparation method for promoting platelet activation of embodiment 4, comprising the following steps:
(1) the fresh yak in Qinghai-tibet skin for taking away 2 centimetres of hairs of skin is cleaned up, shone with the radiation wavelength of 245nm through ultraviolet lamp
It penetrates 0.5 hour, the yak skin after being irradiated.
(2) the yak skin after irradiating is cut into the block of 3cm, extracts in boiling water and takes out after ten minutes, pierces through yak with thorniness plate
Skin.
(3) by step, (2) resulting yak skin is put into the enzymolysis liquid that mass concentration is 4%, is digested 7 hours in 45 DEG C, mistake
Filter, obtains filtrate.
Wherein: the enzymolysis liquid that mass concentration is 4% refers to the addition 40g thiol protease in 1L deionized water, is uniformly mixed
Obtained by solution.
The mass ratio (g/g) of yak skin and enzymolysis liquid is 1:10.
(4) the active carbon of its quality 8% is added in filtrate, is boiled after being sufficiently stirred 10 minutes, filters, respectively obtains for the first time
Digest solution and filter residue.
(5) filter residue is put into the enzymolysis liquid that mass concentration is 7%, is digested 8 hours in 40 DEG C, is obtained second of enzymatic hydrolysis solution.
Wherein: the enzymolysis liquid that mass concentration is 7% refers to the addition 70g bromelain in 1L deionized water, is uniformly mixed
Obtained by solution.
The mass ratio (g/g) of filter residue and enzymolysis liquid is 1:15.
(6) enzymatic hydrolysis solution will digest after solution merges and boil 15 minutes with second for the first time, with the rate of 20000rpm from
The heart separates 45min, obtains supernatant.
(7) supernatant uses aperture for 4.5 μm of hollow-fibre membrane ultrafiltration, obtains Yak-skin Gelatin original protein solution, the yak
Collagen solution in -50 DEG C be freeze-dried 2 hours to get.
A kind of Yak-skin Gelatin preparation method for promoting platelet activation of embodiment 5, comprising the following steps:
(1) the fresh yak in Qinghai-tibet skin for taking away 2 centimetres of hairs of skin is cleaned up, shone with the radiation wavelength of 225nm through ultraviolet lamp
It penetrates 1.5 hours, the yak skin after being irradiated.
(2) the yak skin after irradiating is cut into the block of 1cm, takes out after extracting 25 minutes in boiling water, pierces through yak with thorniness plate
Skin.
(3) by step, (2) resulting yak skin is put into the enzymolysis liquid that mass concentration is 5.5%, is digested 0.5 hour in 40 DEG C,
Filtering, obtains filtrate.
Wherein: the enzymolysis liquid that mass concentration is 5.5% refers in 1L deionized water addition 55g protease, be uniformly mixed and
The solution obtained.Protease refers to 7g bromelain, 26g streptomycete Keratinase, 11g saccharomycete enzyme, 2g pepsin, 4g
Trypsase, 3g cathepsin, 1g papain, 1g subtilopeptidase A mixture.
The mass ratio (g/g) of yak skin and enzymolysis liquid is 1:16.
(4) the active carbon of its quality 7.5% is added in filtrate, is boiled after being sufficiently stirred 10 minutes, filters, respectively obtains first
Secondary enzymatic hydrolysis solution and filter residue.
(5) filter residue is put into the enzymolysis liquid that mass concentration is 8.5%, digests 16 hours in 55 DEG C, it is molten to obtain second of enzymatic hydrolysis
Liquid.
Wherein: the enzymolysis liquid that mass concentration is 8.5% refers in 1L deionized water addition 85g protease, be uniformly mixed and
The solution obtained.Protease refers to the mixture of 39g thiol protease, 14g alkali protease, 32g compound protease.
The mass ratio (g/g) of filter residue and enzymolysis liquid is 1:13.
(6) enzymatic hydrolysis solution will digest after solution merges and boil 20 minutes with second for the first time, with the rate of 18000rpm from
The heart separates 10min, obtains supernatant.
(7) supernatant uses aperture for 6.0 μm of hollow-fibre membrane ultrafiltration, obtains Yak-skin Gelatin original protein solution, the yak
Collagen solution in -35 DEG C be freeze-dried 24 hours to get.
In above-described embodiment 1 ~ 5, protease refers to thiol protease, bromelain, streptomycete Keratinase, yeast
Bacterium enzyme, pepsin, trypsase, cathepsin, papain, subtilopeptidase A, alkali protease, compound egg
The compound of one or both of white enzyme or more.
Claims (10)
1. a kind of Yak-skin Gelatin preparation method for promoting platelet activation, comprising the following steps:
(1) the fresh yak in Qinghai-tibet skin for taking away 1 ~ 2 centimetre of hair of skin is cleaned up, through ultraviolet light irradiation, after being irradiated
Yak skin；
(2) the yak skin after the irradiation is cut into the block of 1 ~ 3cm, takes out after extracting 5 ~ 30 minutes in boiling water, is pierced through with thorniness plate
Yak skin；
(3) by the step, (2) resulting yak skin is put into mass concentration to digest in 0.5% ~ 8% enzymolysis liquid, filters, is filtered
Liquid；The mass ratio of the yak skin and the enzymolysis liquid is 1:5 ~ 1:20；
(4) the active carbon of its quality 3 ~ 10% is added in the filtrate, is boiled after being sufficiently stirred 5 ~ 40 minutes, filters, respectively obtains
Enzymatic hydrolysis solution and filter residue for the first time；
(5) the filter residue is put into the enzymolysis liquid that mass concentration is 1% ~ 10% and digests, and obtains second of enzymatic hydrolysis solution；The filter residue
Mass ratio with the enzymolysis liquid is 1:8 ~ 1:20；
(6) boil 5 ~ 40 minutes, be centrifuged after first time enzymatic hydrolysis solution being merged with second of enzymatic hydrolysis solution,
Obtain supernatant；
(7) the supernatant uses hollow-fibre membrane ultrafiltration, obtains Yak-skin Gelatin original protein solution, the Yak-skin Gelatin original albumen is molten
Liquid it is freeze-dried to get.
2. a kind of Yak-skin Gelatin preparation method for promoting platelet activation as described in claim 1, it is characterised in that: the step
Suddenly (1) middle ultraviolet lamp irradiation condition refers to that radiation wavelength is 200nm ~ 275nm, and irradiation time is 0.3 ~ 2.0 hour.
3. a kind of Yak-skin Gelatin preparation method for promoting platelet activation as described in claim 1, it is characterised in that: the step
Suddenly the enzymolysis liquid that (3) middle mass concentration is 0.5% ~ 8% refers to addition 5 ~ 80g protease in 1L deionized water, is uniformly mixed and obtains
Solution.
4. a kind of Yak-skin Gelatin preparation method for promoting platelet activation as described in claim 1, it is characterised in that: the step
Suddenly (3) middle enzymatic hydrolysis condition refers to that temperature is 30 ~ 60 DEG C, and the time is 0.5 ~ 36 hour.
5. a kind of Yak-skin Gelatin preparation method for promoting platelet activation as described in claim 1, it is characterised in that: the step
Suddenly (5) in mass concentration be 1% ~ 10% enzymolysis liquid refer in 1L deionized water addition 10 ~ 100g protease, be uniformly mixed and
The solution obtained.
6. a kind of Yak-skin Gelatin preparation method for promoting platelet activation as described in claim 1, it is characterised in that: the step
Suddenly (5) middle enzymatic hydrolysis condition refers to that temperature is 30 ~ 55 DEG C, and the time is 0.5 ~ 24 hour.
7. a kind of Yak-skin Gelatin preparation method of promotion platelet activation as claimed in claim 3 or 5, it is characterised in that: institute
It states protease and refers to thiol protease, bromelain, streptomycete Keratinase, saccharomycete enzyme, pepsin, tryptose
One or both of enzyme, cathepsin, papain, subtilopeptidase A, alkali protease, compound protease and
Above compound.
8. a kind of Yak-skin Gelatin preparation method for promoting platelet activation as described in claim 1, it is characterised in that: the step
Suddenly (6) the middle condition being centrifugated refers to that rate is 15000 ~ 25000rpm, and the time is 5 ~ 60min.
9. a kind of Yak-skin Gelatin preparation method for promoting platelet activation as described in claim 1, it is characterised in that: the step
Suddenly (7) the aperture of middle hollow-fibre membrane is 3.5 ~ 7.0 μm.
10. a kind of Yak-skin Gelatin preparation method for promoting platelet activation as described in claim 1, it is characterised in that: described
(7) the middle condition being freeze-dried refers to that temperature is -10 ~ -50 DEG C to step, and the time is 2 ~ 48 hours.
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| CN112385842A (en) * | 2020-12-03 | 2021-02-23 | 中国科学院西北高原生物研究所 | Yak skin glue compound product with coagulation promoting and bleeding stopping effects and preparation method thereof |
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