CN104087641A - Yak skin blood pressure-decreasing collagen polypeptide and preparation method thereof - Google Patents
Yak skin blood pressure-decreasing collagen polypeptide and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a yak skin blood pressure-decreasing collagen polypeptide and a preparation method thereof. The preparation comprises the following steps: pre-treating raw materials by virtue of physical and chemical methods, preparing gelatin, preparing ACE-inhibiting collagen polypeptide by virtue of composite enzymatic hydrolysis, and separating and purifying polypeptide by utilizing methods such as ultra-filtration, ion-exchange column chromatography, gel filtration chromatography and high performance liquid chromatography; concentrating and drying. According to the technical scheme in the invention, collagen polypeptide is successfully extracted from the yak skin, and the collagen polypeptide has good solubility, emulsifying capacity and foaming characteristic, and has higher ACE-inhibiting activity (blood pressure decreasing effect); the preparation method is simple and easy to operate, and low in cost, so that the application prospect of the yak skin is expanded.
Description
Technical field
The present invention relates to a kind of collagen polypeptide and preparation method thereof, more particularly, relate in particular to yak skin blood pressure lowering polypeptide and preparation method thereof.
Background technology
The product that collagen peptide is normally obtained through degradation treatment such as proteolytic enzyme by collagen or gelatin, the amino acid containing and collagen basically identical, particularly feature amino acid oxyproline.The research of modern biological metabolism is found, by a series of enzymic digestion albumen, can be formed some micromolecular materials, and be absorbed by the body.In body, absorption is not only confined to the form of total free aminoacids, and is mainly that the little peptide form forming with 2-3 amino acid directly absorbs.Antihypertensive peptide angiotensin-converting enzyme (ACE) inhibiting peptide that is otherwise known as.It is by competitive inhibition that ACE suppresses polypeptide, hinder the catalyzed reaction of ACE, compare with angiotensin I, the binding ability of ace inhibitory peptide and ACE is stronger, but also be not easy to discharge, thereby hinder the biochemical reaction process that ACE catalytic hydrolysis angiotensin I becomes angiotensinⅡ, the effect that performance reduces blood pressure.
In snake venom, find ace inhibitory peptide, had much research to attempt synthetic ACE and suppressed polypeptide, as enalapril, alacepril and lisinopril etc., these synthetic compounds are used to treat hypertension and the disease such as in heart failure of human body.But synthetic ACE Inhibitor generally all can have side effect, as cough, dysgeusia, fash and vasodilation etc.Therefore, at nearest 10 years, people were striving to find the ACE inhibitor of natural origin, although seldom these inhibitor are free from side effects.
Yak is the important poultry kind of Qinghai-xizang Plateau Region, has indomitable vitality, and growing environment is the Qinghai-Tibet Platean pollution-free, green pure is natural.Each seed amino acid, various nutrient elements, more than the 20 kind of mineral substance useful to human body that in yak skin, contain needed by human.Its chemical composition is mainly comprised of protein, water, inorganic salt, lipid, pigment and carbohydrate etc., wherein moisture accounts for 60%-70%, protein content accounts for 30%-40%, the per-cent of the content of lipid is between 2.0%-4.0%, inorganic salt content is 0.3%-1.0%, and carbohydrate and other account for 0.5%-1.5%.From yak skin, extract natural collagen polypeptide, can widen the kind of yak skin product, thereby make full use of nutritive value and the function of yak skin, avoid waste, protection of the environment.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, yak skin is carried out to collagen protein extraction, carry out extraction and the complex enzyme hydrolysis of gelatin, obtain the collagen peptide with blood pressure reduction effect, the application prospect of expansion yak skin.
Technical purpose of the present invention is achieved by following technical proposals:
The invention provides a kind of preparation method of take the collagen polypeptide with blood pressure reduction effect that yak skin is main raw material, according to above-mentioned preparation method, prepare the hypotensive collagen polypeptide of yak skin, its processing step comprises physics and the pre-treatment of chemical method to raw material, the preparation of gelatin, complex enzyme hydrolysis is prepared ACE and is suppressed collagen polypeptide, by utilizing the methods such as ultrafiltration, ion exchange chromatography, gel permeation chromatography and high performance liquid chromatography to carry out separation and purification polypeptide, concentrate drying, concrete steps are:
Step 1, the depilation of yak skin, soon yak skin is placed in water cleaning and removes surface impurity, after cutting off the hair of giving money as a gift on yak skin, soak yak skin 4～48h in 60～80 ℃ of warm water, machinery thoroughly loses hair or feathers, and clear water rinses, remove soft flocks, skin is cut into the fritter of 0.5 * 0.5cm, obtain the yak skin of depilation, be placed in-18～-20 ℃ of freezing preservations;
Step 2, the degreasing of yak skin, the aqueous solution that the yak skin of soon step 1 depilation is placed in 5wt%～20wt% propyl carbinol is (at solution, the mass percent of propyl carbinol is 5-20%), at solution temperature, be continuously stirring 4～48h at 0～10 ℃, obtain the yak skin of degreasing, the mass ratio of the yak skin of described step 1 depilation and the aqueous solution of propyl carbinol is 1:(5～20);
Step 3, yak skin except foreign protein, the yak skin that is about to step 2 degreasing is placed in alkali lye, continuously stirring 5～48h at 0～10 ℃, obtain the yak skin except foreign protein, wherein said alkali lye is aqueous sodium hydroxide solution or the potassium hydroxide aqueous solution of 0.01～0.2M, and the yak skin of described step 2 degreasing and the mass ratio of alkali lye are 1:(15～40);
Step 4, the depickling of yak skin, be about to step 3 and be placed in acid solution except the yak skin of foreign protein, 20-25 ℃ of room temperatures, stir and soak 2～15h, repeatedly wash again yak skin to pH value and reach 5～7, obtain depickling yak skin, wherein said acid solution is that mass percent is 0.01%～2% aqueous hydrochloric acid or aqueous sulfuric acid, and described step 3 removes the yak skin of foreign protein and the mass ratio of acid solution is 1:(5～15);
Step 5, depickling yak skin is carried out to extracting, the yak skin that is about to step 4 depickling is placed in the hot water of 70～90 ℃ and stirs extracting, the yak skin of described step 4 depickling and the mass ratio of hot water are 1:(5～15), in stirring extractive process, every 30～120min, filter also more heat-exchanging water and proceed to stir extracting, until yak skin all dissolves, each filter obtain become water extract product filtrate gathering;
Step 6, water extract product is prepared to gelatin powder, be about to water extract product (being the whole glues that obtain after yak skin all dissolves) centrifugal 2～20min under 4000～10000g centrifugal force that step 5 obtains, collect supernatant liquor, after utilizing Rotary Evaporators to concentrate, be dried and obtain gelatin powder, 2～4 ℃ store for future use;
Step 7, gelatin powder is carried out to enzymolysis, the gelatin powder that is about to step 6 preparation is dissolved in the buffer solution of sodium phosphate of 0.01～0.2M to obtain the aqueous gelatin solution of mass percent 4～10%, add prozyme to carry out enzymolysis, enzyme concentration is 0.1～2% of substrate (being gelatin powder) weight, hydrolysis temperature is 35～55 ℃, enzymolysis time 4～8h, and pH value is 7; Enzymolysis is placed on 75～90 ℃ of enzymes that go out and processes 5～15min; After deactivation, centrifugal 15～40min under 4000～10000g centrifugal force, retains supernatant liquor;
Step 8, the supernatant liquor of step 7 preparation is carried out to ultrafiltration to obtain the collagen solution of desired molecule amount, wherein choice for use ultra-filtration membrane carries out ultrafiltration, use respectively the ultra-filtration membrane that molecular weight is 10kDa and 3000Da to carry out ultrafiltration, obtaining molecular weight is the collagen solution that is less than 10kDa and 3000Da.
Preferably, in step 2, described propyl carbinol concentration preferably 10-20%, temperature is 5～10 ℃, the time is 20～40h.
Preferably, in step 3, the aqueous solution that described alkali lye is sodium hydroxide, volumetric molar concentration is 0.01M, and removal of impurities temperature is 2～6 ℃, and the time is 15-40h.
Preferably, in step 4, described acid solution is that mass percent is 0.01%～2% aqueous hydrochloric acid, 20-25 ℃ of room temperatures, stirs and soaks 10～15h.
Preferably, in step 6, that after concentrating, carries out is dried as lyophilize or sprays dry.
Preferably, in step 7, complex enzyme zymohydrolysis gelatin, prozyme is trypsinase and Sumizyme MP, both mass ratios are (0.5-1.5): 1, compound protease addition is the 0.1%-2% of gelatin powder quality, enzymolysis 4～8h at 35～55 ℃.
Preferably, after the ultrafiltration of completing steps 8, carry out the methods such as ion exchange chromatography, gel permeation chromatography and high performance liquid chromatography and carry out separation and purification polypeptide, concentrate drying, to obtain the powder of collagen polypeptide.
Utilize technical scheme success of the present invention to extract collagen polypeptide from yak skin, it has good solubleness, emulsifying capacity and bubbling character, and having higher ACE inhibition activity is blood pressure reduction effect.Method for making is simple, easy to operate, cost is low.
Embodiment
Below in conjunction with specific embodiment, further illustrate technical scheme of the present invention.HHL:Hippuryl-Histidyl-Leucin hippuryl Histidine leucine (being purchased from sigma company), the dry yak skin of salt (ox yak market, Qinghai), trypsin analytical reagent, be purchased from Tianjin Novozymes Company), angiotensin-converting enzyme (ACE) analytical pure (being purchased from Tianjin Novozymes Company), Sumizyme MP (Tianjin Novozymes Company provides), spray-drier (Shanghai Ya Cheng company limited).
Example 1
(1) yak skin is placed in to water cleaning and removes surface impurity, cut off again the hair of giving money as a gift on yak skin, after in 80 ℃ of warm water, soak yak skin 20h, machinery is depilation thoroughly, clear water rinses, and removes soft flocks, skin is cut into the fritter of 0.5 * 0.5cm, the yak skin that obtains depilation, is placed in-18～-20 ℃ of freezing preservations;
(2) depilation yak skin is placed in to 12% propyl carbinol, solid-liquid ratio is 1:10, at solution temperature, is continuously stirring 20h at 4 ℃, obtains the yak skin of degreasing;
(3) degreasing yak skin being take to the ratio of 1:15 is placed in the alkali lye that volumetric molar concentration is 0.1M, and continuously stirring 20h at 4 ℃, obtains the yak skin except foreign protein;
(4) will be placed in 0.2% acid solution except foreign protein yak skin, solid-liquid ratio is 1:10, and stirring at room is soaked 5h, more repeatedly washes yak skin to pH value and reach 7, obtains depickling yak skin;
(5) depickling yak skin is placed in to the hot water of 80 ℃ with the ratio of 1:5 and stirs extracting, every 30min, filter and change distilled water, all dissolve to onal, obtain water extract product;
(6) by water extract product, be whole glues, centrifugal 15min under 8000g centrifugal force, collects supernatant liquor;
(7) utilize Rotary Evaporators to concentrate supernatant liquor, rear being dried obtains gelatin powder, and 4 ℃ store for future use;
(8) gelatin powder is dissolved in to the buffer solution of sodium phosphate of 0.1M, obtain 5% gelatin solution, after add prozyme to carry out enzymolysis, enzyme concentration is 1% of substrate weight, prozyme is trypsinase and Sumizyme MP, and both mass ratios are 0.5:1, and hydrolysis temperature is 50 ℃, enzymolysis time 6h, pH value is 7; Enzymolysis is placed on 90 ℃ of enzymes that go out and processes 10min; After deactivation, the centrifugal 30min of 4000g, retains supernatant;
(9) by supernatant liquor, be the ultra-filtration membrane ultrafiltration of 10kDa and 3000Da respectively by molecular weight, obtaining molecular weight is the collagen solution that is less than 10kDa and 3000Da, and ACE suppresses ability IC50 and is respectively 0.802mg/ml, 0.437mg/ml.
Example 2
(1) yak skin is placed in to water cleaning and removes surface impurity, cut off again the hair of giving money as a gift on yak skin, after in 80 ℃ of warm water, soak yak skin 10h, machinery is depilation thoroughly, clear water rinses, and removes soft flocks, skin is cut into the fritter of 0.5 * 0.5cm, the yak skin that obtains depilation, is placed in-18～-20 ℃ of freezing preservations;
(2) depilation yak skin is placed in to 5% propyl carbinol, solid-liquid ratio is 1:5, at solution temperature, is continuously stirring 12h at 4 ℃, obtains the yak skin of degreasing;
(3) degreasing yak skin being take to the ratio of 1:20 is placed in the alkali lye that volumetric molar concentration is 0.1M, and continuously stirring 20h at 5 ℃, obtains the yak skin except foreign protein;
(4) will be placed in 1% acid solution except foreign protein yak skin, solid-liquid ratio is 1:5, and stirring at room is soaked 5h, more repeatedly washes yak skin to pH value and reach 7, obtains depickling yak skin;
(5) depickling yak skin is placed in to the hot water of 80 ℃ with the ratio of 1:5 and stirs extracting, every 40min, filter and change distilled water, all dissolve to onal, obtain water extract product;
(6) by water extract product, be whole glues, centrifugal 15min under 8000g centrifugal force, collects supernatant liquor;
(7) utilize Rotary Evaporators to concentrate supernatant liquor, rear being dried obtains gelatin powder, and 4 ℃ store for future use;
(8) gelatin powder is dissolved in to the buffer solution of sodium phosphate of 0.05M, obtain 4% gelatin solution, after add prozyme to carry out enzymolysis, enzyme concentration is 1% of substrate weight, prozyme is trypsinase and Sumizyme MP, and both mass ratios are 1.5:1, and hydrolysis temperature is 40 ℃, enzymolysis time 6h, pH value is 7; Enzymolysis is placed on 85 ℃ of enzymes that go out and processes 10min; After deactivation, the centrifugal 15min of 8000g, retains supernatant;
(9) by supernatant liquor, be the ultra-filtration membrane ultrafiltration of 10kDa and 3000Da respectively by molecular weight, obtaining molecular weight is the collagen solution that is less than 10kDa and 3000Da, and ACE suppresses ability IC50 and is respectively 4.780mg/ml, 2.012mg/ml.
Example 3
(1) yak skin is placed in to water cleaning and removes surface impurity, cut off again the hair of giving money as a gift on yak skin, after in 60 ℃ of warm water, soak yak skin 4h, machinery is depilation thoroughly, clear water rinses, and removes soft flocks, skin is cut into the fritter of 0.5 * 0.5cm, the yak skin that obtains depilation, is placed in-18～-20 ℃ of freezing preservations;
(2) depilation yak skin is placed in to 10% propyl carbinol, solid-liquid ratio is 1:10, at solution temperature, is continuously stirring 12h at 4 ℃, obtains the yak skin of degreasing;
(3) degreasing yak skin being take to the ratio of 1:40 is placed in the alkali lye that volumetric molar concentration is 0.2M, and continuously stirring 20h at 4 ℃, obtains the yak skin except foreign protein;
(4) will be placed in 2% acid solution except foreign protein yak skin, solid-liquid ratio is 1:5, and stirring at room is soaked 5h, more repeatedly washes yak skin to pH value and reach 6, obtains depickling yak skin;
(5) depickling yak skin is placed in to the hot water of 70 ℃ with the ratio of 1:10 and stirs extracting, every 60min, filter and change distilled water, all dissolve to onal, obtain water extract product;
(6) by water extract product, be whole glues, centrifugal 20min under 6000g centrifugal force, collects supernatant liquor;
(7) utilize Rotary Evaporators to concentrate supernatant liquor, rear being dried obtains gelatin powder, and 4 ℃ store for future use;
(8) gelatin powder is dissolved in to the buffer solution of sodium phosphate of 0.15M, obtain 10% gelatin solution, after add prozyme to carry out enzymolysis, enzyme concentration is 2% of substrate weight, prozyme is trypsinase and Sumizyme MP, and both mass ratios are 0.8:1, and hydrolysis temperature is 35 ℃, enzymolysis time 4h, pH value is 7; Enzymolysis is placed on 75 ℃ of enzymes that go out and processes 10min; After deactivation, the centrifugal 20min of 6000g, retains supernatant;
(9) by supernatant liquor, be the ultra-filtration membrane ultrafiltration of 10kDa and 3000Da respectively by molecular weight, obtaining molecular weight is the collagen solution that is less than 10kDa and 3000Da, and ACE suppresses ability IC50 and is respectively 12.26mg/ml, 5.243mg/ml.
Example 4
(1) yak skin is placed in to water cleaning and removes surface impurity, cut off again the hair of giving money as a gift on yak skin, after in 70 ℃ of warm water, soak yak skin 48h, machinery is depilation thoroughly, clear water rinses, and removes soft flocks, skin is cut into the fritter of 0.5 * 0.5cm, the yak skin that obtains depilation, is placed in-18～-20 ℃ of freezing preservations;
(2) depilation yak skin is placed in to 20% propyl carbinol, solid-liquid ratio is 1:15, at solution temperature, is continuously stirring 12h at 4 ℃, obtains the yak skin of degreasing;
(3) degreasing yak skin being take to the ratio of 1:30 is placed in the alkali lye that volumetric molar concentration is 0.15M, and continuously stirring 48h at 4 ℃, obtains the yak skin except foreign protein;
(4) will be placed in 1.5% acid solution except foreign protein yak skin, solid-liquid ratio is 1:10, and stirring at room is soaked 10h, more repeatedly washes yak skin to pH value and reach 7, obtains depickling yak skin;
(5) depickling yak skin is placed in to the hot water of 90 ℃ with the ratio of 1:10 and stirs extracting, every 80min, filter and change distilled water, all dissolve to onal, obtain water extract product;
(6) by water extract product, be whole glues, centrifugal 5min under 10000g centrifugal force, collects supernatant liquor;
(7) utilize Rotary Evaporators to concentrate supernatant liquor, rear being dried obtains gelatin powder, and 4 ℃ store for future use;
(8) gelatin powder is dissolved in to the buffer solution of sodium phosphate of 0.05M, obtain 4% gelatin solution, after add prozyme to carry out enzymolysis, enzyme concentration is 2% of substrate weight, prozyme is trypsinase and Sumizyme MP, and both mass ratios are 1:1, and hydrolysis temperature is 45 ℃, enzymolysis time 8h, pH value is 7; Enzymolysis is placed on 90 ℃ of enzymes that go out and processes 10min; After deactivation, the centrifugal 15min of 8000g, retains supernatant;
(9) by supernatant liquor, be the ultra-filtration membrane ultrafiltration of 10kDa and 3000Da respectively by molecular weight, obtaining molecular weight is the collagen solution that is less than 10kDa and 3000Da, and ACE suppresses ability IC50 and is respectively 1.67mg/ml, 0.721mg/ml.
Example 5
(1) yak skin is placed in to water cleaning and removes surface impurity, cut off again the hair of giving money as a gift on yak skin, after in 75 ℃ of warm water, soak yak skin 4h, machinery is depilation thoroughly, clear water rinses, and removes soft flocks, skin is cut into the fritter of 0.5 * 0.5cm, the yak skin that obtains depilation, is placed in-18～-20 ℃ of freezing preservations;
(2) depilation yak skin is placed in to 5% propyl carbinol, solid-liquid ratio is 1:5, at solution temperature, is continuously stirring 12h at 10 ℃, obtains the yak skin of degreasing;
(3) degreasing yak skin being take to the ratio of 1:15 is placed in the alkali lye that volumetric molar concentration is 0.15M, and continuously stirring 24h at 10 ℃, obtains the yak skin except foreign protein;
(4) will be placed in 0.05% acid solution except foreign protein yak skin, solid-liquid ratio is 1:5, and stirring at room is soaked 15h, more repeatedly washes yak skin to pH value and reach 7, obtains depickling yak skin;
(5) depickling yak skin is placed in to the hot water of 90 ℃ with the ratio of 1:15 and stirs extracting, every 30min, filter and change distilled water, all dissolve to onal, obtain water extract product;
(6) by water extract product, be whole glues, centrifugal 30min under 4000g centrifugal force, collects supernatant liquor;
(7) utilize Rotary Evaporators to concentrate supernatant liquor, rear being dried obtains gelatin powder, and 4 ℃ store for future use;
(8) gelatin powder is dissolved in to the buffer solution of sodium phosphate of 1M, obtain 5% gelatin solution, after add prozyme to carry out enzymolysis, enzyme concentration is 0.5% of substrate weight, prozyme is trypsinase and Sumizyme MP, and both mass ratios are 1.2:1, and hydrolysis temperature is 55 ℃, enzymolysis time 6h, pH value is 7; Enzymolysis is placed on 90 ℃ of enzymes that go out and processes 10min; After deactivation, the centrifugal 30min of 4000g, retains supernatant;
(9) by supernatant liquor, be the ultra-filtration membrane ultrafiltration of 10kDa and 3000Da respectively by molecular weight, obtaining molecular weight is the collagen solution that is less than 10kDa and 3000Da, and ACE suppresses ability IC50 and is respectively 8.561mg/ml, 2.331mg/ml.
In technical solution of the present invention, the measuring method of ACE inhibition ability is as follows:
HHL substrate solution preparation: HHL is dissolved in to (containing 0.5mol/LNaCl) in pH8.350mmol/L borate buffer, is mixed with the solution of 8.3mmol/L
Get 50 μ l samples and 150 μ lHHL substrate solutions after 37 ℃ of preheating 5min, add 50 μ lACE enzyme liquid (25mU/ml), after 37 ℃ of reaction 60min, add immediately 250 μ l1mol/LHCl termination reactions.The urobenzoic acid that adds 1.5ml ethyl acetate extractive reaction to generate, centrifugal 15min under 1200 * g rotating speed, draws ethyl acetate layer solution 1ml and adds in clean tube, puts into 120 ℃ of solvent flashing 35min of baking oven, finally with 3ml distilled water, dissolve urobenzoic acid, measure the absorbance under 228nm.
A
a: the absorbance that replaces sample with distilled water;
A
b: the absorbance of sample;
A
c: the absorbance that replaces sample and ACE enzyme liquid with distilled water.
Above the present invention has been done to exemplary description; should be noted that; in the situation that not departing from core of the present invention, the replacement that is equal to that any simple distortion, modification or other those skilled in the art can not spend creative work all falls into protection scope of the present invention.
Claims (8)
1. the hypotensive collagen polypeptide of yak skin, is characterized in that, according to following step, carries out:
Step 1, the depilation of yak skin, soon yak skin is placed in water cleaning and removes surface impurity, after cutting off the hair of giving money as a gift on yak skin, soak yak skin 4～48h in 60～80 ℃ of warm water, machinery thoroughly loses hair or feathers, and clear water rinses, remove soft flocks, skin is cut into the fritter of 0.5 * 0.5cm, obtain the yak skin of depilation, be placed in-18～-20 ℃ of freezing preservations;
Step 2, the degreasing of yak skin, the aqueous solution that the yak skin of soon step 1 depilation is placed in 5wt%～20wt% propyl carbinol is (at solution, the mass percent of propyl carbinol is 5-20%), at solution temperature, be continuously stirring 4～48h at 0～10 ℃, obtain the yak skin of degreasing, the mass ratio of the yak skin of described step 1 depilation and the aqueous solution of propyl carbinol is 1:(5～20);
Step 3, yak skin except foreign protein, the yak skin that is about to step 2 degreasing is placed in alkali lye, continuously stirring 5～48h at 0～10 ℃, obtain the yak skin except foreign protein, wherein said alkali lye is aqueous sodium hydroxide solution or the potassium hydroxide aqueous solution of 0.01～0.2M, and the yak skin of described step 2 degreasing and the mass ratio of alkali lye are 1:(15～40);
Step 4, the depickling of yak skin, be about to step 3 and be placed in acid solution except the yak skin of foreign protein, 20-25 ℃ of room temperatures, stir and soak 2～15h, repeatedly wash again yak skin to pH value and reach 5～7, obtain depickling yak skin, wherein said acid solution is that mass percent is 0.01%～2% aqueous hydrochloric acid or aqueous sulfuric acid, and described step 3 removes the yak skin of foreign protein and the mass ratio of acid solution is 1:(5～15);
Step 5, depickling yak skin is carried out to extracting, the yak skin that is about to step 4 depickling is placed in the hot water of 70～90 ℃ and stirs extracting, the yak skin of described step 4 depickling and the mass ratio of hot water are 1:(5～15), in stirring extractive process, every 30～120min, filter also more heat-exchanging water and proceed to stir extracting, until yak skin all dissolves, each filter obtain become water extract product filtrate gathering;
Step 6, water extract product is prepared to gelatin powder, be about to water extract product (being the whole glues that obtain after yak skin all dissolves) centrifugal 2～20min under 4000～10000g centrifugal force that step 5 obtains, collect supernatant liquor, after utilizing Rotary Evaporators to concentrate, be dried and obtain gelatin powder, 2～4 ℃ store for future use;
Step 7, gelatin powder is carried out to enzymolysis, the gelatin powder that is about to step 6 preparation is dissolved in the buffer solution of sodium phosphate of 0.01～0.2M to obtain the aqueous gelatin solution of mass percent 4～10%, add prozyme to carry out enzymolysis, enzyme concentration is 0.1～2% of substrate (being gelatin powder) weight, hydrolysis temperature is 35～55 ℃, enzymolysis time 4～8h, and pH value is 7; Enzymolysis is placed on 75～90 ℃ of enzymes that go out and processes 5～15min; After deactivation, centrifugal 15～40min under 4000～10000g centrifugal force, retains supernatant liquor;
Step 8, the supernatant liquor of step 7 preparation is carried out to ultrafiltration to obtain the collagen solution of desired molecule amount, wherein choice for use ultra-filtration membrane carries out ultrafiltration, use respectively the ultra-filtration membrane that molecular weight is 10kDa and 3000Da to carry out ultrafiltration, obtaining molecular weight is the collagen solution that is less than 10kDa and 3000Da.
2. the hypotensive collagen polypeptide of yak skin according to claim 1, is characterized in that, in step 2, and described propyl carbinol concentration preferably 10-20%, temperature is 5～10 ℃, the time is 20～40h; In step 3, the aqueous solution that described alkali lye is sodium hydroxide, volumetric molar concentration is 0.01M, and removal of impurities temperature is 2～6 ℃, and the time is 15-40h; In step 4, described acid solution is that mass percent is 0.01%～2% aqueous hydrochloric acid, 20-25 ℃ of room temperatures, stirs and soaks 10～15h.
3. the hypotensive collagen polypeptide of yak skin according to claim 1, is characterized in that, in step 6, that after concentrating, carries out is dried as lyophilize or sprays dry.
4. the hypotensive collagen polypeptide of yak skin according to claim 1, it is characterized in that, after the ultrafiltration of completing steps 8, carry out the methods such as ion exchange chromatography, gel permeation chromatography and high performance liquid chromatography and carry out separation and purification polypeptide, concentrate drying, to obtain the powder of collagen polypeptide.
5. the preparation method of the hypotensive collagen polypeptide of yak skin, it is characterized in that, according to following step, carry out: step 1, the depilation of yak skin, soon yak skin is placed in water cleaning and removes surface impurity, after cutting off the hair of giving money as a gift on yak skin, soak yak skin 4～48h in 60～80 ℃ of warm water, machinery thoroughly loses hair or feathers, and clear water rinses, remove soft flocks, skin is cut into the fritter of 0.5 * 0.5cm, obtain the yak skin of depilation, be placed in-18～-20 ℃ of freezing preservations;
Step 2, the degreasing of yak skin, the aqueous solution that the yak skin of soon step 1 depilation is placed in 5wt%～20wt% propyl carbinol is (at solution, the mass percent of propyl carbinol is 5-20%), at solution temperature, be continuously stirring 4～48h at 0～10 ℃, obtain the yak skin of degreasing, the mass ratio of the yak skin of described step 1 depilation and the aqueous solution of propyl carbinol is 1:(5～20);
Step 3, yak skin except foreign protein, the yak skin that is about to step 2 degreasing is placed in alkali lye, continuously stirring 5～48h at 0～10 ℃, obtain the yak skin except foreign protein, wherein said alkali lye is aqueous sodium hydroxide solution or the potassium hydroxide aqueous solution of 0.01～0.2M, and the yak skin of described step 2 degreasing and the mass ratio of alkali lye are 1:(15～40);
Step 4, the depickling of yak skin, be about to step 3 and be placed in acid solution except the yak skin of foreign protein, 20-25 ℃ of room temperatures, stir and soak 2～15h, repeatedly wash again yak skin to pH value and reach 5～7, obtain depickling yak skin, wherein said acid solution is that mass percent is 0.01%～2% aqueous hydrochloric acid or aqueous sulfuric acid, and described step 3 removes the yak skin of foreign protein and the mass ratio of acid solution is 1:(5～15);
Step 5, depickling yak skin is carried out to extracting, the yak skin that is about to step 4 depickling is placed in the hot water of 70～90 ℃ and stirs extracting, the yak skin of described step 4 depickling and the mass ratio of hot water are 1:(5～15), in stirring extractive process, every 30～120min, filter also more heat-exchanging water and proceed to stir extracting, until yak skin all dissolves, each filter obtain become water extract product filtrate gathering;
Step 6, water extract product is prepared to gelatin powder, be about to water extract product (being the whole glues that obtain after yak skin all dissolves) centrifugal 2～20min under 4000～10000g centrifugal force that step 5 obtains, collect supernatant liquor, after utilizing Rotary Evaporators to concentrate, be dried and obtain gelatin powder, 2～4 ℃ store for future use;
Step 7, gelatin powder is carried out to enzymolysis, the gelatin powder that is about to step 6 preparation is dissolved in the buffer solution of sodium phosphate of 0.01～0.2M to obtain the aqueous gelatin solution of mass percent 4～10%, add prozyme to carry out enzymolysis, enzyme concentration is 0.1～2% of substrate (being gelatin powder) weight, hydrolysis temperature is 35～55 ℃, enzymolysis time 4～8h, and pH value is 7; Enzymolysis is placed on 75～90 ℃ of enzymes that go out and processes 5～15min; After deactivation, centrifugal 15～40min under 4000～10000g centrifugal force, retains supernatant liquor;
Step 8, the supernatant liquor of step 7 preparation is carried out to ultrafiltration to obtain the collagen solution of desired molecule amount, wherein choice for use ultra-filtration membrane carries out ultrafiltration, use respectively the ultra-filtration membrane that molecular weight is 10kDa and 3000Da to carry out ultrafiltration, obtaining molecular weight is the collagen solution that is less than 10kDa and 3000Da.
6. the preparation method of the hypotensive collagen polypeptide of yak skin according to claim 5, is characterized in that, in step 2, and described propyl carbinol concentration preferably 10-20%, temperature is 5～10 ℃, the time is 20～40h; In step 3, the aqueous solution that described alkali lye is sodium hydroxide, volumetric molar concentration is 0.01M, and removal of impurities temperature is 2～6 ℃, and the time is 15-40h; In step 4, described acid solution is that mass percent is 0.01%～2% aqueous hydrochloric acid, 20-25 ℃ of room temperatures, stirs and soaks 10～15h.
7. the preparation method of the hypotensive collagen polypeptide of yak skin according to claim 5, is characterized in that, in step 6, that after concentrating, carries out is dried as lyophilize or sprays dry.
8. the preparation method of the hypotensive collagen polypeptide of yak skin according to claim 5, it is characterized in that, after the ultrafiltration of completing steps 8, carry out the methods such as ion exchange chromatography, gel permeation chromatography and high performance liquid chromatography and carry out separation and purification polypeptide, concentrate drying, to obtain the powder of collagen polypeptide.
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