CN106148466A - A kind of preparation method of yak collagen protein - Google Patents
A kind of preparation method of yak collagen protein Download PDFInfo
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- CN106148466A CN106148466A CN201610781871.0A CN201610781871A CN106148466A CN 106148466 A CN106148466 A CN 106148466A CN 201610781871 A CN201610781871 A CN 201610781871A CN 106148466 A CN106148466 A CN 106148466A
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 36
- 108010035532 Collagen Proteins 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 23
- 239000012530 fluid Substances 0.000 claims abstract description 22
- 229920001436 collagen Polymers 0.000 claims abstract description 16
- 239000007787 solid Substances 0.000 claims abstract description 5
- 239000004365 Protease Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 13
- 238000010298 pulverizing process Methods 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 12
- 238000004587 chromatography analysis Methods 0.000 claims description 12
- 229940088598 enzyme Drugs 0.000 claims description 11
- 108090000526 Papain Proteins 0.000 claims description 9
- 108090000284 Pepsin A Proteins 0.000 claims description 9
- 102000057297 Pepsin A Human genes 0.000 claims description 9
- 102000004142 Trypsin Human genes 0.000 claims description 9
- 108090000631 Trypsin Proteins 0.000 claims description 9
- 229940055729 papain Drugs 0.000 claims description 9
- 235000019834 papain Nutrition 0.000 claims description 9
- 229940111202 pepsin Drugs 0.000 claims description 9
- 239000012588 trypsin Substances 0.000 claims description 9
- 210000000003 hoof Anatomy 0.000 claims description 8
- 210000004761 scalp Anatomy 0.000 claims description 8
- 229960001322 trypsin Drugs 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 7
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 235000019419 proteases Nutrition 0.000 claims description 5
- 108090000145 Bacillolysin Proteins 0.000 claims description 2
- 102000035092 Neutral proteases Human genes 0.000 claims description 2
- 108091005507 Neutral proteases Proteins 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 6
- 210000001519 tissue Anatomy 0.000 description 46
- 239000012528 membrane Substances 0.000 description 20
- 239000000243 solution Substances 0.000 description 17
- 210000004379 membrane Anatomy 0.000 description 13
- 239000013078 crystal Substances 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 102000009027 Albumins Human genes 0.000 description 7
- 108010088751 Albumins Proteins 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 229940081735 acetylcellulose Drugs 0.000 description 6
- 229920002301 cellulose acetate Polymers 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 230000007935 neutral effect Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 5
- 102000002322 Egg Proteins Human genes 0.000 description 5
- 108010000912 Egg Proteins Proteins 0.000 description 5
- 239000003513 alkali Substances 0.000 description 5
- 235000014103 egg white Nutrition 0.000 description 5
- 210000000969 egg white Anatomy 0.000 description 5
- 230000002255 enzymatic effect Effects 0.000 description 5
- 238000001976 enzyme digestion Methods 0.000 description 5
- 238000012856 packing Methods 0.000 description 5
- 238000001223 reverse osmosis Methods 0.000 description 5
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 description 4
- 102000004882 Lipase Human genes 0.000 description 4
- 108090001060 Lipase Proteins 0.000 description 4
- 239000004367 Lipase Substances 0.000 description 4
- 102000035195 Peptidases Human genes 0.000 description 4
- 102000015439 Phospholipases Human genes 0.000 description 4
- 108010064785 Phospholipases Proteins 0.000 description 4
- 235000019421 lipase Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 241000209094 Oryza Species 0.000 description 3
- 235000007164 Oryza sativa Nutrition 0.000 description 3
- 239000004952 Polyamide Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000004760 aramid Substances 0.000 description 3
- 229920003235 aromatic polyamide Polymers 0.000 description 3
- 238000010612 desalination reaction Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 229920002647 polyamide Polymers 0.000 description 3
- 235000009566 rice Nutrition 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 241000283074 Equus asinus Species 0.000 description 2
- 240000005001 Paeonia suffruticosa Species 0.000 description 2
- 235000003889 Paeonia suffruticosa Nutrition 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 238000007598 dipping method Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 235000013601 eggs Nutrition 0.000 description 2
- 239000003205 fragrance Substances 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229920001007 Nylon 4 Polymers 0.000 description 1
- 229920002292 Nylon 6 Polymers 0.000 description 1
- 229920002302 Nylon 6,6 Polymers 0.000 description 1
- 239000004695 Polyether sulfone Substances 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- SMEGJBVQLJJKKX-HOTMZDKISA-N [(2R,3S,4S,5R,6R)-5-acetyloxy-3,4,6-trihydroxyoxan-2-yl]methyl acetate Chemical compound CC(=O)OC[C@@H]1[C@H]([C@@H]([C@H]([C@@H](O1)O)OC(=O)C)O)O SMEGJBVQLJJKKX-HOTMZDKISA-N 0.000 description 1
- 238000005377 adsorption chromatography Methods 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 108010052008 colla corii asini Proteins 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 229940042795 hydrazides for tuberculosis treatment Drugs 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920006393 polyether sulfone Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- 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
-
- 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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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- General Engineering & Computer Science (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses the preparation method of a kind of yak collagen protein.The preparation method of the yak collagen protein of the present invention, first pulverizes yak tissue, then enzymolysis, then separates then purification from the tissue fluid that enzymolysis obtains, obtain collagen solids, so that the preparation method of the present invention is relatively simple.
Description
Technical field
The present invention relates to the technical field of collagen protein, in particular to the preparation method of a kind of yak collagen protein.
Background technology
Collagen protein is biopolymer, is the functional protein that mammal in-vivo content is most, distribution is the widest, accounts for egg
The 25%～30% of white matter total amount, some organism is even as high as more than 80%.Collagen protein is because having good bio-compatible
Property, biodegradable and biological activity, therefore obtain in fields such as food, medicine, organizational project, cosmetics and widely should
With.The collagen health product " Colla Corii Asini " of such as Chinese tradition, being mainly with donkey skin as raw material (is exactly contained collagen egg in donkey skin in fact
In vain), there is higher nourishing health function, have the good reputation of one of Chinese medicine three essentials-essence.
In prior art, complex with the method that yak produces collagen protein for raw material.
Summary of the invention
In view of this, one aspect of the present invention is to provide the preparation method of a kind of yak collagen protein, and this preparation method is relatively
For simply.
The preparation method of a kind of yak collagen protein, comprises the following steps:
(1) yak tissue is pulverized；
(2) by carrying out enzymolysis through the yak tissue of step (1), tissue fluid is obtained；
(3) collagen liquid in separating from described tissue fluid；
(4) described collagen liquid is chromatographed, obtain collagen solids.
Further, described yak tissue one in yak flaw-piece, yak hoof, yak scalp and Os Bovis grunniens or extremely
Few two kinds.
Further, described pulverizing uses tissue pulverizer, and the rotating speed of tissue pulverizer is 3000～5000rpm, pulverizes
Temperature be-10～0 DEG C, the time of pulverizing is 5～15min.
Further, the protease that described enzymolysis is used is selected from pepsin, trypsin, papain and neutrality
One in protease or at least two kinds.
Further, the temperature of described enzymolysis is-10～0 DEG C.
Further, described separate from described tissue fluid in the mode of collagen liquid be centrifugal.
Further, described centrifugal rotating speed is 11000～13000rpm, and the centrifugal time is 1～10min.
Further, the pressure of chromatography is 0.08～0.12MPa, and the temperature of chromatography is-10～0 DEG C
Further, also include after described chromatography being dried, described dry employing lyophilization.
Further, described dry temperature is-70～-60 DEG C.
Further, also including being dried after described chromatography, described dry temperature is-70～60 DEG C.
The preparation method of the yak collagen protein of the present invention, first pulverizes yak tissue, then enzymolysis, then obtains from enzymolysis
To tissue fluid in separate then purification, obtain collagen solids, so that the preparation method of the present invention is relatively simple.
Detailed description of the invention
For the ease of understanding the present invention, close embodiment below and further illustrate technical scheme.
As used herein, term:
" one ", " a kind of " and " described " are used interchangeably and refer to one or more.
"and/or" for represent the one or both of illustrated situation all it may happen that, such as, A and/or B includes (A
And B) and (A or B)；
It addition, all numerical value that the scope stated by end points herein is comprised in the range of including this (such as, 1 to 10 bag
Include 1.4,1.9,2.33,5.75,9.98 etc.).
It addition, the statement of " at least one " herein include one and above all numbers (such as, at least 2, at least
4, at least 6, at least 8, at least 10, at least 25, at least 50, at least 100 etc.).
The preparation method of the yak collagen protein of the present invention, comprises the following steps:
(1) yak tissue is pulverized；
(2) by carrying out enzymolysis through the yak tissue of step (1), tissue fluid is obtained；
(3) collagen liquid in separating from described tissue fluid；
(4) by described collagen liquid purification, collagen solids is obtained
Above-mentioned yak tissue can be connective tissue, such as, be retrieved from yak flaw-piece, yak hoof, yak scalp and Os Bovis grunniens
In one or its combination in any.
For obtaining the purity of higher collagen protein, yak tissue can also be carried out before step (1) described pulverizing
Ungrease treatment.Herein, the effect of ungrease treatment is to remove the fat in yak tissue.The mode of defat can use alkali
Liquid, alkali liquor has sodium hydroxide solution, potassium hydroxide solution, sodium bicarbonate solution or its any mixed liquor here.Employing alkali liquor takes off
Fat can use the mode of dipping, and the consumption of alkali liquor is 2～3 times of yak liver mass, such as 2 times, 2.2 times, 2.5 times, 2.8
Again, 2.9 times or 3 times.The mass concentration of alkali liquor is 0.5～10wt%, such as 0.5wt%, 0.55wt%, 0.6wt%, 1wt%,
2wt%, 5wt%, 5.5wt%, 6wt%, 8wt%, 9wt%, 9.5wt%, 9.8wt% or 10wt% etc..The temperature of dipping can
At room temperature, it is preferably 25～35 DEG C, such as 25 DEG C, 26 DEG C, 28 DEG C, 30 DEG C, 32 DEG C, 33 DEG C, 34 DEG C or 35 DEG C etc..Leaching
The time of stain can be 12～60h, such as 12h, 13h, 15h, 20h, 24h, 30h, 36h, 48h, 54h, 58h or 60h etc..
Ungrease treatment, except using the mode of soda-dip, also can use phospholipase and lipase to carry out enzymolysis.
The conversion ratio of enzymolysis is higher.The concrete kind of lipase and phospholipase is not particularly limited to by the present invention, such as, can use Tianjin
Nuo Ao enzyme preparation company limited is produced.
The consumption of phospholipase and lipase does not do the most harsh regulation, but preferably, phospholipase can be to account for treat defat
The 0.3～3 ‰ of oil Paeonia suffruticosa dregs of rice quality, such as 0.3 ‰, 0.5 ‰, 1 ‰, 1.5 ‰, 2 ‰, 2.5 ‰, 2.8 ‰ or 3 ‰.Lipase
Can account for treat 0.2～2 ‰, such as the 0.2 ‰ of oily Paeonia suffruticosa dregs of rice quality of defat, 0.5 ‰, 1 ‰, 1.5 ‰, 1.8 ‰, 1.9 ‰ or
0.2‰.These two kinds of enzymes are too much, will not bring significantly improving of conversion ratio；Cross the conversion ratio that can affect lipid at least.
The preferable temperature of the enzymolysis of defat is 30～40 DEG C, such as 30 DEG C, 32 DEG C, 35 DEG C, 38 DEG C, 39 DEG C or 40 DEG C.
Under the temperature premise of above-mentioned enzyme dosage and enzymolysis, the time of enzymolysis is preferably 0.5～5h, such as 0.5h, 0.75h, 1h,
2h, 3h, 4h, 4.5h, 5h or 5h etc..
The mode of above-mentioned pulverizing can use tissue pulverizer.The concrete type of tissue pulverizer can use known model,
Do not describe in detail in this.The rotating speed of tissue pulverizer is preferred with 3000～5000rpm, such as 3000rpm, 3100rpm, 3500rpm,
4000rpm, 4500rpm, 4800rpm, 4900rpm or 5000rpm etc., preferably 3800rpm.
Pulverize temperature be preferably-10～0 DEG C, such as-10 DEG C ,-9 DEG C ,-8 DEG C ,-5 DEG C ,-4 DEG C ,-3 DEG C ,-2 DEG C ,-
1 DEG C or 0 DEG C etc..At a temperature of this, the time of pulverizing is preferably 5～15min, such as 5min, 6min, 8min, 10min,
11min, 13min, 14min, 15min etc., preferably 10min etc..
Above-mentioned enzymolysis is carried out in the case of adding protease.Herein, protease is the class of enzymes of aminosal peptide chain
General name.Enzymolysis protein can list pepsin, trypsin, papain and neutral protease and its any group
Close.The pH of enzymolysis can select according to the concrete kind of these enzymes, such as, for pepsin, the pH of enzymolysis can be 1～2；Right
In trypsin, the pH of enzymolysis can be 7.5～8.5；For papain, the pH of enzymolysis can be 6～7；For neutral protein
Enzyme, the pH of enzymolysis can be 6.8～7.0.
The temperature of enzymolysis preferably-10～0 DEG C, such as-10 DEG C ,-9 DEG C ,-8 DEG C ,-5 DEG C ,-4 DEG C ,-3 DEG C ,-2 DEG C ,-
1 DEG C or 0 DEG C etc., the time as enzymolysis can use the time generally in the art, or according to combining actual adjusting.
In separating from described tissue fluid, the mode of collagen liquid can list centrifugal, membrance separation, dialysis process, preferably
It is centrifugal.Here, it is centrifuged and is i.e. centrifuged precipitating or centrifugation, refer to produce at a relatively high angle speed by equipment such as centrifuges
Degree, makes centrifugal force be much larger than gravity, and then the float in solution is convenient to Precipitation: again due to material institute that proportion is different
The centrifugal force being subject to is different, thus sedimentation velocity is different, and the material that proportion can be made different reaches to separate.Membrance separation refers to utilize film
Reach to separate to the crown_interception of macromole.Membrance separation can use ultrafilter membrane (UF) and reverse osmosis membrane (RO).Ultrafilter membrane is a kind of
Aperture specification is consistent, and nominal pore scope is the micropore filtering film of less than 0.01 micron.Suitable pressure is imposed, just in the side of film
The solute molecule less than aperture can be sifted out, receive more than 10 more than 500 dalton (atomic mass unit), particle diameter separating molecular weight
The granule of rice.Its operation principle is: with the pressure differential of film both sides as driving force, with ultrafilter membrane as filter medium, in certain pressure
Under power, when stock solution flows through film surface, the tiny micropore of many that ultrafiltration membrane surface gathers only allows water and small-molecule substance to lead to
Crossing and become permeate, in stock solution, volume is then trapped within the liquid feeding side of film more than the material in film surface micropore footpath, becomes dense
Contracting liquid, thus the purpose realizing the purification to stock solution, separating and concentrate.There are about 6,000,000,000 on the ultrafiltration membrane filaments tube wall of every meter long
The micropore of 0.01 micron, its aperture only allows the beneficial mineral matter in hydrone, water and trace element to pass through, and smallest bacteria
Volume all more than 0.02 micron, therefore antibacterial and the colloid more much bigger than antibacterial volume, rust, float, silt, divide greatly
Sub-Organic substances etc. can be retained down by ultrafilter membrane, it is achieved thereby that purification process.Ultrafilter membrane both can use inoranic membrane, it is possible to
To use organic membrane, inoranic membrane can list ceramic membrane and metal film；Organic membrane can list cellulose acetate, aromatic series polyamides
The instantiation of amine, polyether sulfone, Kynoar etc..
Reverse osmosis membrane is also known as reverse osmosis, a kind of with pressure differential as motive force, isolates the membrance separation behaviour of solvent from solution
Make.The feed liquid of film side is applied pressure, and when pressure exceedes its osmotic pressure, solvent can be made anti-against the direction of naturally osmotic
To infiltration.Thus the solvent passed through, i.e. penetrating fluid is obtained in the low-pressure side of film；High-pressure side obtains the solution concentrated, i.e. concentrated solution.
Reverse osmosis membrane can be cellulose acetate, and cellulose acetate is also known as acetylcellulose or cellulose acetate, often with cellulose
Cotton Gossypii, timber etc. be raw material, make cellulose acetate through over-churning and hydrolysis, be reprocessed into reverse osmosis membrane, or can
Fatty polyamide and aromatic polyamide is included, such as nylon 4, nylon 6, Nylon 66 Membrane, fragrance adoption for polyamide
Amide film.Membrane material is aromatic polyamide, aromatic polyamide hydrazides and some nitrogenous aromatic polymers, fragrance adoption
The pH scope that amide film adapts to can be wide to 2～11, but very sensitive to the free chlorine in water.Or can be composite membrane, it refers to
Both the above material is made, and it is to be composited with the thinnest compacted zone and porous support layer.Porous support layer, also known as basement membrane, rises
Strengthen the effect of mechanical strength；Compacted zone is also referred to as epidermal area, plays desalination, therefore also known as desalination layer.Desalination layer thickness can be
30～50nm.Herein, dialysis refer to be diffused into through semi-permeable membrane the principle of water (or buffer) by little molecule, by little molecule with
The separate a kind of separate mode of biomacromolecule.Dialysis can use bag filter to implement.
Above-mentioned centrifugal rotating speed can be 11000～13000rpm, such as 11000rpm, 11100rpm, 11500rpm,
12000rpm, 12500rpm, 12800rpm, 13000rpm, preferably 12000rpm.Under this rotating speed, the centrifugal time can be 1
～10min, such as 1min, 1.5min, 2min, 3min, 5min, 6min, 8min, 9min, 9.5min or 10min etc., it is preferably
5min。
The mode of above-mentioned purification is chromatography.Herein, protein chromatographic refers to utilize the difference of protein physical property, will be many
Component mixture separates.Chromatography can use gel filtration, ion-exchange chromatography, adsorption chromatography and affinity chromatograph etc..Layer
Analysis can use albumin layer analyzer.Albumin layer analyzer can use conventional model.
Chromatography pressure be advisable with 0.08～0.12MPa, such as 0.08MPa, 0.085MPa, 0.09MPa, 0.10MPa,
0.11MPa, 0.115MPa, 0.118MPa, 0.119MPa or 0.12MPa etc., preferably 0.10MPa.Under the pressure of this precipitating,
The temperature of chromatography is-10～0 DEG C, such as-10 DEG C ,-9 DEG C ,-8 DEG C ,-5 DEG C ,-4 DEG C ,-3 DEG C ,-2 DEG C ,-1 DEG C or 0 DEG C etc..
Also include after chromatography being dried.The mode being dried can use lyophilization.Herein, lyophilization is dry also known as distillation
Dry, refer to be chilled to water-containing materials below freezing, make water be changed into ice, then under high vacuum, ice is changed into steam and
Remove.In the present invention, lyophilization can use vacuum lyophilization.Vacuum lyophilization by wet stock or solution in relatively low temperature
It is frozen into solid-state under degree (-10 DEG C～-50 DEG C), then makes moisture therein direct without liquid under vacuum (1.3～13Pa)
It is sublimed into gaseous state, finally makes material dewatering.Certainly, it is dried and also can use mode known to other, such as spray drying etc., omit in this
State.
Above-mentioned cryodesiccated temperature can be-70～-60 DEG C, such as-70 DEG C ,-69 DEG C ,-68 DEG C ,-65 DEG C ,-64 DEG C ,-
63 DEG C ,-62 DEG C ,-61 DEG C or-60 DEG C etc..
Embodiment 1
Step one, by yak tissue (such as yak flaw-piece, yak hoof, yak scalp, Os Bovis grunniens etc.) clean after, put into group
Knit and pulverizer (rotating speed 3000rpm rpm, temperature-10 DEG C) carries out tissue pulverizing, pulverize 15min, must organize unqualified.
Step 2, it is placed in unqualified for tissue in enzymatic vessel, adds pepsin, trypsin, papain, neutral egg
White enzyme carries out enzyme digestion reaction, obtains tissue fluid (temperature controls at-10 DEG C)；
Step 3, tissue fluid is placed in low-temperature and high-speed centrifuge (rotating speed 11000rpm, temperature-10 DEG C) centrifugal, centrifugal
10min, obtains supernatant, and filtering residue is separately used.
Step 4, supernatant is placed in albumin layer analyzer (pressure 0.08MPa, temperature-10 DEG C), obtains chromatographic solution.
Step 5, chromatographic solution is placed in freeze dryer it is dried (temperature-70 DEG C), obtain lyophilizing crystal.
Step 6, by crystals weighed, be loaded on respectively in packing container, obtain yak collagen protein finished product.
Embodiment 2
Step one, by yak tissue (such as yak flaw-piece, yak hoof, yak scalp, Os Bovis grunniens etc.) clean after, put into group
Knit and pulverizer (rotating speed 5000rpm rpm, temperature 0 DEG C) carries out tissue pulverizing, pulverize 5min, must organize unqualified.
Step 2, it is placed in unqualified for tissue in enzymatic vessel, adds pepsin, trypsin, papain, neutral egg
White enzyme carries out enzyme digestion reaction, obtains tissue fluid (temperature controls at 0 DEG C)；
Step 3, tissue fluid is placed in low-temperature and high-speed centrifuge (rotating speed 13000rpm, temperature-10 DEG C) centrifugal, centrifugal
1min, obtains supernatant, and filtering residue is separately used.
Step 4, supernatant is placed in albumin layer analyzer (pressure 0.08MPa, temperature-10 DEG C), obtains chromatographic solution.
Step 5, chromatographic solution is placed in freeze dryer it is dried (temperature-70 DEG C), obtain lyophilizing crystal.
Step 6, by crystals weighed, be loaded on respectively in packing container, obtain yak collagen protein finished product.
Embodiment 3
Step one, by yak tissue (such as yak flaw-piece, yak hoof, yak scalp, Os Bovis grunniens etc.) clean after, put into group
Knit and pulverizer (rotating speed 5000rpm rpm, temperature-10 DEG C) carries out tissue pulverizing, pulverize 5min, must organize unqualified.
Step 2, it is placed in unqualified for tissue in enzymatic vessel, adds pepsin, trypsin, papain, neutral egg
White enzyme carries out enzyme digestion reaction, obtains tissue fluid (temperature controls at 0 DEG C)；
Step 3, tissue fluid is placed in low-temperature and high-speed centrifuge (rotating speed 113000rpm, temperature 0 DEG C) centrifugal, centrifugal
1min, obtains supernatant, and filtering residue is separately used.
Step 4, supernatant is placed in albumin layer analyzer (pressure 0.12MPa, temperature 0 DEG C), obtains chromatographic solution.
Step 5, chromatographic solution is placed in freeze dryer it is dried (temperature-60 DEG C), obtain lyophilizing crystal.
Step 6, by crystals weighed, be loaded on respectively in packing container, obtain yak collagen protein finished product.
Embodiment 4
Step one, by yak tissue (such as yak flaw-piece, yak hoof, yak scalp, Os Bovis grunniens etc.) clean after, put into group
Knit and pulverizer (rotating speed 4000rpm, temperature-5 DEG C) carries out tissue pulverizing, pulverize 5min, must organize unqualified.
Step 2, it is placed in unqualified for tissue in enzymatic vessel, adds pepsin, trypsin, papain, neutral egg
White enzyme carries out enzyme digestion reaction, obtains tissue fluid (temperature controls at 0 DEG C)；
Step 3, tissue fluid is placed in low-temperature and high-speed centrifuge (rotating speed 12000rpm, temperature-5 DEG C) centrifugal, centrifugal
5min, obtains supernatant, and filtering residue is separately used.
Step 4, supernatant is placed in albumin layer analyzer (pressure 0.010MPa, temperature 0 DEG C), obtains chromatographic solution.
Step 5, chromatographic solution is placed in freeze dryer it is dried (temperature-70 DEG C), obtain lyophilizing crystal.
Step 6, by crystals weighed, be loaded on respectively in packing container, obtain yak collagen protein finished product.
Embodiment 5
Step one, by yak tissue (such as yak flaw-piece, yak hoof, yak scalp, Os Bovis grunniens etc.) clean after, put into group
Knit and pulverizer (rotating speed 4000rpm rpm, temperature-5 DEG C) carries out tissue pulverizing, pulverize 10min, must organize unqualified.
Step 2, it is placed in unqualified for tissue in enzymatic vessel, adds pepsin, trypsin, papain, neutral egg
White enzyme carries out enzyme digestion reaction, obtains tissue fluid (temperature controls at-5 DEG C)；
Step 3, tissue fluid is placed in low-temperature and high-speed centrifuge (rotating speed 12000rpm, temperature-5 DEG C) centrifugal, centrifugal
5min, obtains supernatant, and filtering residue is separately used.
Step 4, supernatant is placed in albumin layer analyzer (pressure 0.10MPa, temperature-5 DEG C), obtains chromatographic solution.
Step 5, chromatographic solution is placed in freeze dryer it is dried (temperature-65 DEG C), obtain lyophilizing crystal.
Step 6, by crystals weighed, be loaded on respectively in packing container, obtain yak collagen protein finished product.
Owing to the numerical range of each technological parameter involved in the present invention can not all embody in the above-described embodiments,
As long as but the most envisioned any numerical value fallen in this numerical range above-mentioned of those skilled in the art all can implement this
Invention, the most also includes the combination in any of occurrence in the range of some numerical value.Herein, for the consideration of length, eliminate to
Going out the embodiment of occurrence in certain one or more numerical range, this disclosure being not to be construed as technical scheme is not filled
Point.
Applicant states, the present invention illustrates detailed process equipment and the technological process of the present invention by above-described embodiment,
But the invention is not limited in above-mentioned detailed process equipment and technological process, i.e. do not mean that the present invention have to rely on above-mentioned in detail
Process equipment and technological process could be implemented.Person of ordinary skill in the field it will be clearly understood that any improvement in the present invention,
The equivalence of raw material each to product of the present invention is replaced and the interpolation of auxiliary element, concrete mode selection etc., falls in the protection of the present invention
In the range of.
Claims (10)
1. the preparation method of a yak collagen protein, it is characterised in that comprise the following steps:
(1) yak tissue is pulverized；
(2) by carrying out enzymolysis through the yak tissue of step (1), tissue fluid is obtained；
(3) collagen liquid in separating from described tissue fluid；
(4) described collagen liquid is chromatographed, obtain collagen solids.
Preparation method the most according to claim 1, it is characterised in that described yak tissue selected from yak flaw-piece, yak hoof,
One in yak scalp and Os Bovis grunniens or at least two kinds.
Preparation method the most according to claim 1, it is characterised in that described pulverizing uses tissue pulverizer, tissue to pulverize
The rotating speed of machine is 3000～5000rpm, and the temperature of pulverizing is-10～0 DEG C, and the time of pulverizing is 5～15min.
Preparation method the most according to claim 1, it is characterised in that the protease that described enzymolysis is used is selected from pepsin
One in enzyme, trypsin, papain and neutral protease or at least two kinds.
Preparation method the most according to claim 1, it is characterised in that the temperature of described enzymolysis is-10～0 DEG C.
6. according to preparation method as claimed in claim 1, it is characterised in that described from described tissue fluid separate in collagen egg
The mode of white liquor is centrifugal.
7. according to preparation method as claimed in claim 6, it is characterised in that described centrifugal rotating speed be 11000～
13000rpm, the centrifugal time is 1～10min.
8. according to preparation method as claimed in claim 1, it is characterised in that the pressure of described chromatography is 0.08～0.12MPa,
The temperature of chromatography is-10～0 DEG C.
9. according to preparation method as claimed in claim 1, it is characterised in that also include after described chromatography being dried, described dry
Dry employing lyophilization.
10. according to preparation method as claimed in claim 1, it is characterised in that described dry temperature is-70～-60 DEG C.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701878A (en) * | 2017-02-20 | 2017-05-24 | 青海师范大学 | Method for extracting yak skin collagen by adopting enzyme process |
| CN107354192A (en) * | 2017-08-18 | 2017-11-17 | 广东医科大学 | A kind of method for purifying NTx albumen |
| CN107630061A (en) * | 2017-09-29 | 2018-01-26 | 中国农业科学院农产品加工研究所 | The preparation method of Yak Bone NTx albumen |
| CN113201065A (en) * | 2021-04-19 | 2021-08-03 | 国肽生物工程（常德）有限公司 | Bovine bone collagen peptide with functions of relieving fatigue and improving bone density and preparation method thereof |
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| CN103468771A (en) * | 2013-05-27 | 2013-12-25 | 河北考力森生物科技有限公司 | Method for extracting collagens from bovine achilles tendon |
| CN105385737A (en) * | 2015-12-09 | 2016-03-09 | 青海北极牦牛生物科技有限公司 | Preparation technology of yak bone collagen oligopeptide |
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| CN103468771A (en) * | 2013-05-27 | 2013-12-25 | 河北考力森生物科技有限公司 | Method for extracting collagens from bovine achilles tendon |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106701878A (en) * | 2017-02-20 | 2017-05-24 | 青海师范大学 | Method for extracting yak skin collagen by adopting enzyme process |
| CN107354192A (en) * | 2017-08-18 | 2017-11-17 | 广东医科大学 | A kind of method for purifying NTx albumen |
| CN107630061A (en) * | 2017-09-29 | 2018-01-26 | 中国农业科学院农产品加工研究所 | The preparation method of Yak Bone NTx albumen |
| CN113201065A (en) * | 2021-04-19 | 2021-08-03 | 国肽生物工程（常德）有限公司 | Bovine bone collagen peptide with functions of relieving fatigue and improving bone density and preparation method thereof |
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