CN105385739B - - kind of the method that protein peptides are produced from golden-rimmed leech - Google Patents
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- 241000545744 Hirudinea Species 0.000 title claims abstract description 107
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 38
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 34
- 102000004169 proteins and genes Human genes 0.000 title claims description 13
- 108090000623 proteins and genes Proteins 0.000 title claims description 13
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 77
- 238000001914 filtration Methods 0.000 claims abstract description 61
- 238000001471 micro-filtration Methods 0.000 claims abstract description 47
- 239000003513 alkali Substances 0.000 claims abstract description 34
- 229920001184 polypeptide Polymers 0.000 claims abstract description 31
- 230000000694 effects Effects 0.000 claims abstract description 24
- 230000001376 precipitating effect Effects 0.000 claims abstract description 18
- 238000001694 spray drying Methods 0.000 claims abstract description 11
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 238000001728 nano-filtration Methods 0.000 claims abstract description 6
- 230000001954 sterilising effect Effects 0.000 claims abstract description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims description 90
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 52
- 239000000243 solution Substances 0.000 claims description 46
- 238000003756 stirring Methods 0.000 claims description 36
- 108090000790 Enzymes Proteins 0.000 claims description 34
- 102000004190 Enzymes Human genes 0.000 claims description 34
- 229940088598 enzyme Drugs 0.000 claims description 34
- 238000010792 warming Methods 0.000 claims description 34
- 239000008367 deionised water Substances 0.000 claims description 26
- 229910021641 deionized water Inorganic materials 0.000 claims description 26
- 239000004744 fabric Substances 0.000 claims description 26
- 239000002893 slag Substances 0.000 claims description 26
- 239000012466 permeate Substances 0.000 claims description 22
- 239000000706 filtrate Substances 0.000 claims description 20
- 239000012141 concentrate Substances 0.000 claims description 17
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 claims description 15
- 239000002002 slurry Substances 0.000 claims description 15
- 238000007654 immersion Methods 0.000 claims description 14
- 230000008569 process Effects 0.000 claims description 13
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 12
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 11
- 229910052708 sodium Inorganic materials 0.000 claims description 11
- 239000011734 sodium Substances 0.000 claims description 11
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 10
- 229920001661 Chitosan Polymers 0.000 claims description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 108010019160 Pancreatin Proteins 0.000 claims description 10
- 229940055695 pancreatin Drugs 0.000 claims description 10
- 239000012460 protein solution Substances 0.000 claims description 10
- 239000000758 substrate Substances 0.000 claims description 10
- 239000012286 potassium permanganate Substances 0.000 claims description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 238000009413 insulation Methods 0.000 claims description 7
- 229960000583 acetic acid Drugs 0.000 claims description 6
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- 239000004677 Nylon Substances 0.000 claims description 5
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical class [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 claims description 5
- 235000010323 ascorbic acid Nutrition 0.000 claims description 5
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- 238000010257 thawing Methods 0.000 claims description 5
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- 238000010438 heat treatment Methods 0.000 claims 1
- 239000002253 acid Substances 0.000 abstract description 14
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- 102000007625 Hirudins Human genes 0.000 description 17
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- WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 description 16
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- 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 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 4
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- 241000500851 Poecilobdella manillensis Species 0.000 description 4
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- 239000007921 spray Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
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- 239000011149 active material Substances 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
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- 210000003079 salivary gland Anatomy 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 241000243818 Annelida Species 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 241000237858 Gastropoda Species 0.000 description 1
- 108010003272 Hyaluronate lyase Proteins 0.000 description 1
- 102000001974 Hyaluronidases Human genes 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000237536 Mytilus edulis Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000570011 Pomacea canaliculata Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000002585 base Substances 0.000 description 1
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- 238000001727 in vivo Methods 0.000 description 1
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- OTQCKZUSUGYWBD-BRHMIFOHSA-N lepirudin Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)C(C)C)[C@@H](C)O)[C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(C)C)[C@@H](C)O)C1=CC=C(O)C=C1 OTQCKZUSUGYWBD-BRHMIFOHSA-N 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to technical field of bioengineering, and in particular to a kind of method that polypeptide is produced from golden-rimmed leech.A kind of method that polypeptide is produced from golden-rimmed leech, comprises the following steps：Sterilization, defibrination, coarse filtration, micro-filtration, ultrafiltration, alkali soluble, microwave treatment, centrifugation, precipitating, decolouring, enzymolysis, precipitating, micro-filtration, ultrafiltration, nanofiltration, concentration and spray drying.Offer of the present invention substitutes chemical method separation Acid polypeptide with physical method, obtains Acid polypeptide crude product, can compound the Chinese patent drug containing Acid polypeptide or make the preceding extract of purifying Acid polypeptide; separation rate is high; its activity can be protected very well, it is pollution-free to produce, reach clean manufacturing.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to a kind of method that protein peptides are produced from golden-rimmed leech.
Background technology
Leech also known as leech, belong to annelid Hirudinea, and body surface is without bristle, and there is a sucker front and back end, and body flesh is flourishing, body cavity compared with
It is small, belong to the annelid of eggcase.It is reported that leech in the world is divided into by feeding habits and sucked blood and nonbloodsucking two more than 500 kinds
Major class, the leech overwhelming majority is lived in fresh water, and salt water and salt-fresh water intersection are also found, and also has a small number of lives by land,
In moist spinney and thick grass that mountain area is lived in if the mountain leech sucked blood, at home, individual larger is eurysome golden thread leech,
Body is up to 30cm, and weigh 40g, but finds a kind of appearance like golden-rimmed leech in the Ruili mountain area in Yunnan recently, and body weight reaches 150g, individual
Body minimum is that few leech only has 0.1g.
Leech is different according to its feeding habits, can be divided into nonbloodsucking leech and the major class of hirudinaria manillensis two：
Nonbloodsucking leech, it mainly using the molluscan juice such as river snail, Pomacea canaliculata, freshwater mussel as food, do not suck blood, main product in
River, the lake in province (such as Hubei, Jiangsu) along the Yangtze River.Commonly eurysome golden thread leech (Whitmanpigra
Whitman), it is the main flow kind in China's medicinal material market, its in vivo containing at present not yet clearly it is some it is promoting blood circulation and removing blood stasis into
Point, but not hirudin.
Hirudinaria manillensis, is mainly distributed on the provinces and regions such as Guangdong, the Guangxi in China south China, lives in rice field, ditch, rivers, pond
In the pool and lake, made a living with sucking people and vertebrate blood, wherein relatively conventional is hiruto
(PoecilobdellaManillensis Lesson, are commonly called as golden-rimmed leech, and it is supervised by Guangxi Zhuang Autonomous Region food and medicine
Superintend and direct management board include into《Strengthen medicine quality standard in Guangxi Zhuang Autonomous Region》Volume Two (version in 2011).Scientific investigations showed that, by inhaling
In the salivary gland of watery blood leech and its secreted saliva containing abundant hirudin hirudin, hyaluronidase Orgelase with
And the various bioactivators such as vasodilator Vasolilator and anesthetic Anaesthetic, wherein hirudin is these
Quantity is at most in active material, activity is most notable and most studied, most deep, most have medical value and most development potentiality
A kind of active material.
Natural hirudin, in the 70-80 ages in 20th century, just enters from isolating after sterling to its physicochemical property and chemical constitution
Further investigation is gone, result of study shows, the polypeptide thing that hirudin is made up of 65-66 amino acid residue, molecular weight is
There are 3 disulphide bridgeses at 7000DaN ends, are made up of 6 half Guang ammonia, N-terminal peptide chain is rich in acid ammonia around cyclic peptide structures, C-terminal is changed into
Base acid residue, has hydrophily, is free in the middle part of molecular surface, peptide chain and also has one by Pro-Lys47-Pro (proline-rely ammonia
Acid) composition special sequence, it is acidic amino acid to have 5 in last 9 amino acid residues of C-terminal, therefore also known as Acid polypeptide, this
The outer tyrosine for occuping 63 is sulphated.After the composition and structure sequence of natural hirudin are understood, just start to artificial
Lepirudin 023 ludon carries out the research of nearly 10 years, obtains that product is extremely similar to natural hirudin, only occupy the junket of 63 in sequence
Propylhomoserin fails sulphation, during products application its suppress the active relatively low of fibrin ferment and cause bleeding and the list defect such as target spot, make
With narrow range, side effect is big, cost is high etc., thus application is extremely restricted.
There are some researches show hirudin is primarily present in the salivary gland and saliva of leech of sucking blood, the leech saliva do not sucked blood
Hirudin composition is can't check in gland and saliva.The different kind in the leech sucked blood, different regions, Individual Size is wild
Influence is produced on leech cellulose content with the factor such as picking time of cultivation leech.At present, find to originate in Guangdong, Guangxi, Hainan, cloud
The leech cellulose content of the wild golden-rimmed leech (being commonly called as ox leech) of 21-23 ° of strip of parallel of north latitude of the provinces such as south is higher, more
The highest content caught after winter reaches 1200ATU/g (the butt meter of whole piece dry product), is caught in by the end of May, 2015 in Beibu Bay, guangxi
The leech cellulose content caught also reaches 1020ATU/g.
The method of traditional extraction hirudin is after hirudinaria manillensis living or chilled leech thaw, to cut head or all defibrinations,
Extractant is made of alcohol grading precipitating or of acetone, hirudin solution of crude product is obtained, then using chromatography, isoelectric focusing etc.
Process is purified, and obtains hirudin.These processes are not directed to extract after hirudin, the utilization of a large amount of protein, and conventional
Method, using a large amount of solvents, there is certain risk in the control of its contaminative and security.
The information for being disclosed in the background section is merely intended to understanding of the increase to the general background of the present invention, without answering
When the prior art for being considered as recognizing or implying the information structure in any form well known to persons skilled in the art.
The content of the invention
In order to overcome the deficiencies in the prior art, the invention provides a kind of method that protein peptides are produced from golden-rimmed leech.
In order to solve the above technical problems, the present invention uses following technical scheme：
A kind of method that polypeptide is produced from golden-rimmed leech, comprises the following steps：
(1) pollutant of fresh leech or defrosting leech body surface is first rinsed with clear water, concentration is then moved into for 3 ‰ potassium permanganate
In solution, stirring immersion 10-15min；Wash liquor potassic permanganate after picking up off with clear water, then move it into concentration for 5% chlorination
Stirring immersion 10min, washes away the salt of its body surface with clear water in sodium solution after picking up, then with deionized water rinsing 1 time, drains water
Point, it is placed in container；
(2) by after leech body surface sterilization, calculated by leech weight, add 3 ‰ sodium hydrogensulfites, 1 ‰ ascorbic acid
Mix thoroughly, ground in input colloid mill, grinding temperature is controlled less than 50 DEG C, obtains leech slurries with 3 times of deionized waters；
(3) the leech slurries obtained by step (2) are subjected to 2 coarse filtration, the filter residue of 2 coarse filtration of merging and filtrate；
(4) filtrate obtained by step (3) is subjected to micro-filtration with 30000 films that can intercept molecular weight 30000Da, collects micro-filtration saturating
Cross liquid；
(5) micro-filtration permeate is subjected to 2 ultrafiltration, 10000 films are selected in the 1st ultrafiltration；Concentrate adds above-mentioned coarse filtration slag
In, permeate carries out the 2nd ultrafiltration, from 5000 films；It is incorporated to through solution in foregoing filter residue, gained dope is that enrichment contains
Hirudin solution；
(6) coarse filtration slag and micro-filtration concentrate and the dope of ultrafiltration 1, the transparent liquid of ultrafiltration 2, it is 9 to adjust pH value, and input has stirring to fill
Put, in power 2-10KW microwave tank, heated up with 2KW power microwaves, control temperature control is less than 60 DEG C, microwave 20min, at 55-60 DEG C
It is incubated 20min, discharge cooling；
(7) solution for discharging step (6) is cooled to 45 DEG C, is filtered, is accumulated in filter cloth with 200 mesh filter cloth tripod pendulum type batch centrifugals
Filter residue is sprayed in right amount with deionized water, and gained filtrate is leech protein solution；
(8) by leech protein solution obtained by step (7), pH value first is adjusted to 6 with watery hydrochloric acid, it is then molten with 10% glacial acetic acid again
The chitosan solution of solution 2% adjusts pH value to 5, and albumen is separated out to come, 1-2h is stood, and is used again after discarding upper liquid after solution layering
400 mesh filter cloth centrifugal filtrations, obtain wet albumen, add the 2-3 times of wet liquor potassic permanganate of protein content 0.75% in 35-45 DEG C of temperature
Lower immersion 1.5-2h, potassium permanganate is then sloughed with 400 mesh nylon filtering cloths and is rinsed 1 time with clear water, with 2-3 times of wet slag
0.75% oxalic acid solution soaks 2-3h, when observation albumen color is white, that is, starts to collect decolouring albumen；
(9) the wet albumen of decolouring is diluted with deionized water, when protein concentration reaches 7%, it is 9 to adjust pH value, is added
The alkaline enzyme of substrate quality 3% stirs, and is placed in band agitating device power 2-10KW microwave tanks, starts 2KW power, work as enzyme
Solution liquid detects enzymolysis liquid pH value when being warming up to 45 DEG C, when pH value is dropped to below more than 7.5 8, mends alkali and is adjusted to 8.5, enzymolysis liquid
It is warming up to 55 DEG C and examines 1 pH value again, alkali is no longer mended when pH is declined by less than less than 7.8, alkali is mended when dropping to less than 7.8 and is adjusted to 8, enzyme
Solution liquid temperature degree stops microwave when reaching 55 DEG C and retains stirring, restarts 2KW power microwaves after temperature drop is less than 50 DEG C and treats enzyme
Solution liquid is warming up to 55 DEG C, turns off microwave and retains stirring, enzymolysis liquid pH value is detected when enzymolysis liquid temperature drop is to less than 45 DEG C, when
Alkali is mended when pH is less than 8 and is adjusted to 8, the pancreatin of addition substrate quality 2.5% starts 2KW microwaves, when enzymolysis liquid is warming up to 50 DEG C, inspection
Enzymolysis liquid pH value is looked into, when pH is descend below or during close to 7, alkali is mended and is adjusted to 7.8, continues microwave and be warming up to 55 DEG C to turn off microwave, then
PH is detected, 7.5 is adjusted to when pH drops to less than 7.3 benefit alkali, starts stirring, treats that enzymolysis liquid temperature drop restarts micro- work(to 45 DEG C
Rate 2KW, enzymolysis liquid is warming up to 55 DEG C, after being incubated 5 minutes, and startup power 10KW is warming up to 85-90 DEG C, is incubated 10 minutes enzymes that go out
It is living；
(10) enzymolysis liquid after enzyme activity of going out is discharged, is cooled to 40 DEG C, under stirring, be slowly added with containing 20% ice
The chitosan solution of acetate dissolution 1.5%, makes pH value be adjusted to 5, albumen that precipitating is not digested and other big molecular impurities etc., precipitating
Liquid stands 2 hours, and the precipitating thing being centrifuged off with 400 mesh filter clothes is leech albumen, first filters and is filtered after upper liquid during centrifugation
Dope；
(11) filtrate obtained by 400 mesh are filtered, then carry out micro-filtration with 30000 films；Dope is merged into above-mentioned 400 mesh coarse filtration
In slag；
(12) micro-filtration permeate obtained by step (11) carries out 2 ultrafiltration, and the 1st 10000 films of ultrafiltration, the 2nd ultrafiltration is used
5000 films, the concentrate of two times of ultrafiltration is merged into coarse filtration slag；
(13) the gained permeate of ultrafiltration 2 in step (12) is subjected to sodium filter；
(14) by concentrate obtained by nanofiltration in step (13), dried from spray drying process, obtain polypeptide；By step (10)
Filter residue, the dope of step (11) micro-filtration, the dope of step (12) ultrafiltration merge to be dried from spray drying process, obtains leech egg
White powder.
In technical scheme, the fineness of gained leech slurries is 18-20 mesh in step (2).
In technical scheme, the concrete operations of 2 coarse filtration described in step (3) are：1st coarse filtration 60-
80 mesh filter clothes are filtered in tripod pendulum type batch centrifugal, and the filter residue accumulated in filter cloth is sprayed 1-2 times with appropriate deionized water；2nd time coarse filtration is used
200 mesh are disembarked filtering in tripodia, and the filter residue accumulated in filter cloth is sprayed 1-2 times with appropriate deionized water.
In technical scheme, the operating parameter of micro-filtration is in step (4)：Press pump 0.05-0.3MPa, pH value 6-7,
40-50 DEG C of temperature, film surface flow 1-4M/S, 3-4 times of cocnentration factor.
In technical scheme, the operating parameter of the 1st ultrafiltration described in step (5) is：Press pump 0.1-
0.43MPa, pH value 5-6,40-50 DEG C of temperature, crossflow velocity 1-3M/S, 4-5 times of cocnentration factor；The operating parameter of 2nd ultrafiltration is：
Press pump 0.3-0.6MPa, pH value are 5-6,40-50 DEG C of temperature, film surface flow 1-3M/S, 6-7 times of cocnentration factor.
In technical scheme, the enzyme activity of the alkaline enzyme described in step (9) is 100,000 u/g；The enzyme activity of pancreatin>
4000u/g。
In technical scheme, the operating parameter of micro-filtration is in step (11)：Press pump 0.05-0.3MPa, pH value 6-
7th, 40-50 DEG C of temperature, crossflow velocity 1-4M/S, 3-4 times of cocnentration factor.
In technical scheme, the operating parameter of the 1st ultrafiltration and the 2nd ultrafiltration is in step (12)：Press pump
0.2-0.4MPa, pH value 5-6,40-50 DEG C of temperature, crossflow velocity 1-3M/S, 5-6 times of cocnentration factor.
In technical scheme, the operating parameter of the filter of sodium described in step (13) is：Pressure 0.7-0.9MPa, circulation
Flow 1.5-2.5m3/ h, cycles of concentration 7-9.
Compared with prior art, the present invention has the advantages that：
1. offer of the present invention substitutes chemical method separation Acid polypeptide with physical method, Acid polypeptide crude product is obtained, can be answered
Preceding extract with the Chinese patent drug containing Acid polypeptide or work purifying Acid polypeptide, separation rate is high, and its activity can be protected very well, pollution-free
Produce, reach clean manufacturing.
2. the golden-rimmed leech that uses of method of the present invention is high containing protein, isolate still suffered from after Acid polypeptide it is very high
Protein, is digested, and is obtained based on small peptide, containing only a small amount of free amino acid and the mixture of small point of albumen, than
The protein adsorption not digested is more faster, and physiological function is stronger.
3. the method for the present invention also obtains leech albumen powder except acquisition Acid polypeptide crude product, leech polypeptide mixture,
Leech skin for producing chitin, makes the exploitation of leech reach preliminary comprehensive utilization.
Brief description of the drawings
Fig. 1 produces the process chart of polypeptide for the present invention from golden-rimmed leech.
Embodiment
With reference to specific embodiment, make further details of elaboration to the present invention, but embodiments of the present invention are not
It is confined to the scope that embodiment is represented.These embodiments are merely to illustrate the present invention, not for limitation the scope of the present invention.This
Outside, after present disclosure is read, those skilled in the art can various modifications may be made to the present invention, and these equivalent variations are same
Sample falls within appended claims limited range of the present invention.
Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments
Material, reagent for using etc., unless otherwise specified, are commercially obtained.
Embodiment 1：
A kind of method that polypeptide is produced from golden-rimmed leech, comprises the following steps：
(1) pollutant of fresh leech or defrosting leech body surface is first rinsed with clear water, concentration is then moved into for 3 ‰ potassium permanganate
In solution, stirring immersion 10min；Wash liquor potassic permanganate after picking up off with clear water, then it is that 5% sodium chloride is molten to move it into concentration
Stirring immersion 10min, washes away the salt of its body surface with clear water in liquid after picking up, then with deionized water rinsing 1 time, drains moisture,
It is placed in container；
(2) by after leech body surface sterilization, calculated by leech weight, add 3 ‰ sodium hydrogensulfites, 1 ‰ ascorbic acid
Mix thoroughly, ground in input colloid mill, grinding temperature is controlled less than 50 DEG C, obtains leech slurries with 3 times of deionized waters；Described
The fineness of leech slurries is 18 mesh；
(3) the leech slurries obtained by step (2) are subjected to 2 coarse filtration, the filter residue of 2 coarse filtration of merging and filtrate；Described 2
The concrete operations of secondary coarse filtration are：1st coarse filtration is filtered with 60 mesh filter clothes in tripod pendulum type batch centrifugal, accumulates the filter residue in filter cloth with right amount
Deionized water is sprayed 1 time；2nd coarse filtration is disembarked filtering, the appropriate deionized water of filter residue accumulated in filter cloth with 200 mesh in tripodia
Spray 1 time；
(4) filtrate obtained by step (3) is subjected to micro-filtration with 30000 films that can intercept molecular weight 30000Da, collects micro-filtration saturating
Cross liquid；The operating parameter of micro-filtration is：Press pump 0.05Mpa, pH value 6,40 DEG C of temperature, film surface flow 1M/S, 3 times of cocnentration factor；
(5) micro-filtration permeate is subjected to 2 ultrafiltration, 10000 films are selected in the 1st ultrafiltration；Concentrate adds above-mentioned coarse filtration slag
In, permeate carries out the 2nd ultrafiltration, from 5000 films；It is incorporated to through solution in foregoing filter residue, gained dope is that enrichment contains
Hirudin solution；The operating parameter of the 1st time described ultrafiltration is：Press pump 0.1Mpa, pH value 5,40 DEG C of temperature, crossflow velocity 1M/
4 times of S, cocnentration factor；The operating parameter of 2nd ultrafiltration is：Press pump 0.3Mpa, pH value be 5,40 DEG C of temperature, film surface flow 1M/S, dense
Contract 6 times of ratio；
(6) coarse filtration slag and micro-filtration permeate and the dope of ultrafiltration 1, the transparent liquid of ultrafiltration 2, it is 9 to adjust pH value, and input has stirring to fill
Put, in power 2KW microwave tank, heated up with 2KW power microwaves, control temperature control is less than 60 DEG C, microwave 20min, in 55 DEG C of insulations
20min, discharge cooling；
(7) solution for discharging step (6) is cooled to 45 DEG C, is filtered, is accumulated in filter cloth with 200 mesh filter cloth tripod pendulum type batch centrifugals
Filter residue is sprayed in right amount with deionized water, and gained filtrate is leech protein solution；
(8) by leech protein solution obtained by step (7), pH value first is adjusted to 6 with watery hydrochloric acid, it is then molten with 10% glacial acetic acid again
The chitosan solution of solution 2% adjusts pH value to 5, and albumen is separated out to come, 1h is stood, and is used again after discarding upper liquid after solution layering
400 mesh filter cloth centrifugal filtrations, obtain wet albumen, add 2 times of wet liquor potassic permanganates of protein content 0.75% and are soaked at a temperature of 35 DEG C
1.5h is steeped, potassium permanganate is then sloughed with 400 mesh nylon filtering cloths and is rinsed 1 time with clear water, with 0.75% oxalic acid of 2 times of wet slags
Solution soaks 2h, when observation albumen is white, that is, starts to collect decolouring albumen；
(9) the wet albumen of decolouring is diluted with deionized water, when protein concentration reaches 7%, it is 9 to adjust pH value, is added
The alkaline enzyme of substrate quality 3% stirs, and is placed in band agitating device power 2KW microwave tanks, starts 2KW power, work as enzymolysis
Liquid detects enzymolysis liquid pH value when being warming up to 45 DEG C, when pH value is dropped to below 7.5, mends alkali lye and is adjusted to 9, when pH value drops to
When less than more than 7.5 8, mend alkali and be adjusted to 8.5, enzymolysis liquid is warming up to 55 DEG C and examines 1 pH value again, when pH is declined by less than less than 7.8 not
Alkali is mended again, and alkali is mended when dropping to less than 7.8 and is adjusted to 8, enzymolysis liquid temperature stops microwave when reaching 55 DEG C and retains stirring, at a temperature for the treatment of
It is reduced to after 50 DEG C and restarts 2KW power microwaves and treat that enzymolysis liquid is warming up to 55 DEG C, turns off microwave and retain stirring, treat enzymolysis liquid temperature
Enzymolysis liquid pH value is detected when dropping to less than 45 DEG C, mending alkali when pH is less than 8 is adjusted to 8, adds the pancreatin of substrate quality 2.5%,
Start 2KW microwaves, when enzymolysis liquid is warming up to 50 DEG C, check enzymolysis liquid pH value, when pH is descend below or during close to 7, mend alkali and be adjusted to
7.8, continuation microwave is warming up to 55 DEG C and turns off microwave, then detects pH, is adjusted to 7.5 when pH drops to less than 7.3 benefit alkali, startup is stirred
Mix, treat that enzymolysis liquid temperature drop restarts micropower 2KW to 45 DEG C, enzymolysis liquid is warming up to 55 DEG C, after insulation 5 minutes, start work(
Rate 10KW is warming up to 85 DEG C, is incubated 10 minutes enzyme activity of going out；The enzyme activity of described alkaline enzyme is 100,000 u/g；The enzyme activity of pancreatin>
4000u/g；
(10) enzymolysis liquid after enzyme activity of going out is discharged, is cooled to 40 DEG C, under stirring, be slowly added with containing 20% ice
The chitosan solution of acetate dissolution 1.5%, makes pH value be adjusted to 5, albumen that precipitating is not digested and other big molecular impurities etc., precipitating
Liquid stands 2 hours, and the precipitating thing being centrifuged off with 400 mesh filter clothes is leech albumen, first filters and is filtered after upper liquid during centrifugation
Dope；
(11) filtrate obtained by 400 mesh are filtered, then carry out micro-filtration with 30000 films；Dope is merged into above-mentioned 400 mesh coarse filtration
In slag；The operating parameter of micro-filtration is：Press pump 0.05Mpa, pH value 6,40 DEG C of temperature, crossflow velocity 1M/S, 3 times of cocnentration factor；
(12) micro-filtration permeate obtained by step (11) carries out 2 ultrafiltration, and the 1st 10000 films of ultrafiltration, the 2nd ultrafiltration is used
5000 films, the concentrate of two times of ultrafiltration is merged into coarse filtration slag；The operating parameter of 1st ultrafiltration and the 2nd ultrafiltration is：Press pump
0.2Mpa, pH value 5,40 DEG C of temperature, crossflow velocity 1M/S, 5 times of cocnentration factor；
(13) the gained permeate of ultrafiltration 2 in step (12) is subjected to sodium filter；The operating parameter of sodium filter is：Pressure
0.7Mpa, circular flow 1.5m3/ h, cycles of concentration 7.
(14) by concentrate obtained by nanofiltration in step (13), dried from spray drying process, obtain polypeptide；By step (10)
Filter residue, the dope of step (11) micro-filtration, the dope of step (12) ultrafiltration merge to be dried from spray drying process, obtains leech egg
White powder.
Embodiment 2：
A kind of method that polypeptide is produced from golden-rimmed leech, comprises the following steps：
(1) pollutant of fresh leech or defrosting leech body surface is first rinsed with clear water, concentration is then moved into for 3 ‰ potassium permanganate
In solution, stirring immersion 15min；Wash liquor potassic permanganate after picking up off with clear water, then it is that 5% sodium chloride is molten to move it into concentration
Stirring immersion 10min, washes away the salt of its body surface with clear water in liquid after picking up, then with deionized water rinsing 1 time, drains moisture,
It is placed in container；
(2) by after leech body surface sterilization, calculated by leech weight, add 3 ‰ sodium hydrogensulfites, 1 ‰ ascorbic acid
Mix thoroughly, ground in input colloid mill, grinding temperature is controlled less than 50 DEG C, obtains leech slurries with 3 times of deionized waters；Described
The fineness of leech slurries is 20 mesh；
(3) the leech slurries obtained by step (2) are subjected to 2 coarse filtration, the filter residue of 2 coarse filtration of merging and filtrate；Described 2
The concrete operations of secondary coarse filtration are：1st coarse filtration is filtered with 80 mesh filter clothes in tripod pendulum type batch centrifugal, accumulates the filter residue in filter cloth with right amount
Deionized water is sprayed 2 times；2nd coarse filtration is disembarked filtering, the appropriate deionized water of filter residue accumulated in filter cloth with 200 mesh in tripodia
Spray 2 times；
(4) filtrate obtained by step (3) is subjected to micro-filtration with 30000 films that can intercept molecular weight 30000Da, collects micro-filtration saturating
Cross liquid；The operating parameter of micro-filtration is：Press pump 0.3Mpa, pH value 7, temperature 50 C, film surface flow 4M/S, 4 times of cocnentration factor；
(5) micro-filtration permeate is subjected to 2 ultrafiltration, 10000 films are selected in the 1st ultrafiltration；Concentrate adds above-mentioned coarse filtration slag
In, permeate carries out the 2nd ultrafiltration, from 5000 films；It is incorporated to through solution in foregoing filter residue, gained dope is that enrichment contains
Hirudin solution；The operating parameter of the 1st time described ultrafiltration is：Press pump 0.43Mpa, pH value 6, temperature 50 C, crossflow velocity 3M/
5 times of S, cocnentration factor；The operating parameter of 2nd ultrafiltration is：Press pump 0.6Mpa, pH value be 6, temperature 50 C, film surface flow 3M/S, dense
Contract 7 times of ratio；
(6) coarse filtration slag and micro-filtration permeate and the dope of ultrafiltration 1, the transparent liquid of ultrafiltration 2, it is 9 to adjust pH value, and input has stirring to fill
Put, in power 10KW microwave tank, heated up with 2KW power microwaves, control temperature control is less than 60 DEG C, microwave 20min, in 60 DEG C of insulations
20min, discharge cooling；
(7) solution for discharging step (6) is cooled to 45 DEG C, is filtered, is accumulated in filter cloth with 200 mesh filter cloth tripod pendulum type batch centrifugals
Filter residue is sprayed in right amount with deionized water, and gained filtrate is leech protein solution；
(8) by leech protein solution obtained by step (7), pH value first is adjusted to 6 with watery hydrochloric acid, it is then molten with 10% glacial acetic acid again
The chitosan solution of solution 2% adjusts pH value to 5, and albumen is separated out to come, 2h is stood, and is used again after discarding upper liquid after solution layering
400 mesh filter cloth centrifugal filtrations, obtain wet albumen, add 3 times of wet liquor potassic permanganates of protein content 0.75% and are soaked at a temperature of 45 DEG C
2h is steeped, potassium permanganate is then sloughed with 400 mesh nylon filtering cloths and is rinsed 1 time with clear water, 0.75% oxalic acid with 3 times of wet slags is molten
Immersion steeps 3h, when observation albumen is white, that is, starts to collect decolouring albumen；
(9) the wet albumen of decolouring is diluted with deionized water, when protein concentration reaches 7%, it is 9 to adjust pH value, is added
The alkaline enzyme of substrate quality 3% stirs, and is placed in band agitating device power 10KW microwave tanks, starts 2KW power, work as enzymolysis
Liquid detects enzymolysis liquid pH value when being warming up to 45 DEG C, when pH value is dropped to below 7.5, mends alkali lye and is adjusted to 9, when pH value drops to
When less than more than 7.5 8, mend alkali and be adjusted to 8.5, enzymolysis liquid is warming up to 55 DEG C and examines 1 pH value again, when pH is declined by less than less than 7.8 not
Alkali is mended again, and alkali is mended when dropping to less than 7.8 and is adjusted to 8, enzymolysis liquid temperature stops microwave when reaching 55 DEG C and retains stirring, at a temperature for the treatment of
It is reduced to after 50 DEG C and restarts 2KW power microwaves and treat that enzymolysis liquid is warming up to 55 DEG C, turns off microwave and retain stirring, treat enzymolysis liquid temperature
Enzymolysis liquid pH value is detected when dropping to less than 45 DEG C, mending alkali when pH is less than 8 is adjusted to 8, adds the pancreatin of substrate quality 2.5%,
Start 2KW microwaves, when enzymolysis liquid is warming up to 50 DEG C, check enzymolysis liquid pH value, when pH is descend below or during close to 7, mend alkali and be adjusted to
7.8, continuation microwave is warming up to 55 DEG C and turns off microwave, then detects pH, is adjusted to 7.5 when pH drops to less than 7.3 benefit alkali, startup is stirred
Mix, treat that enzymolysis liquid temperature drop restarts micropower 2KW to 45 DEG C, enzymolysis liquid is warming up to 55 DEG C, after insulation 5 minutes, start work(
Rate 10KW is warming up to 85-90 DEG C, is incubated 10 minutes enzyme activity of going out；The enzyme activity of described alkaline enzyme is 100,000 u/g；The enzyme activity of pancreatin>
4000u/g；
(10) enzymolysis liquid after enzyme activity of going out is discharged, is cooled to 40 DEG C, under stirring, be slowly added with containing 20% ice
The chitosan solution of acetate dissolution 1.5%, makes pH value be adjusted to 5, albumen that precipitating is not digested and other big molecular impurities etc., precipitating
Liquid stands 2 hours, and the precipitating thing being centrifuged off with 400 mesh filter clothes is leech albumen, first filters and is filtered after upper liquid during centrifugation
Dope；
(11) filtrate obtained by 400 mesh are filtered, then carry out micro-filtration with 30000 films；Dope is merged into above-mentioned 400 mesh coarse filtration
In slag；The operating parameter of micro-filtration is：Press pump 0.3Mpa, pH value 7, temperature 50 C, crossflow velocity 4M/S, 4 times of cocnentration factor；
(12) micro-filtration permeate obtained by step (11) carries out 2 ultrafiltration, and the 1st 10000 films of ultrafiltration, the 2nd ultrafiltration is used
5000 films, the concentrate of two times of ultrafiltration is merged into coarse filtration slag；The operating parameter of 1st ultrafiltration and the 2nd ultrafiltration is：Press pump
0.4Mpa, pH value 6, temperature 50 C, crossflow velocity 3M/S, 6 times of cocnentration factor；
(13) the gained permeate of ultrafiltration 2 in step (12) is subjected to sodium filter；The operating parameter of sodium filter is：Pressure
0.9Mpa, circular flow 2.5m3/ h, cycles of concentration 9.
(14) by concentrate obtained by nanofiltration in step (13), dried from spray drying process, obtain polypeptide；By step (10)
Filter residue, the dope of step (11) micro-filtration, the dope of step (12) ultrafiltration merge to be dried from spray drying process, obtains leech egg
White powder.
Embodiment 3：
A kind of method that polypeptide is produced from golden-rimmed leech, comprises the following steps：
(1) pollutant of fresh leech or defrosting leech body surface is first rinsed with clear water, concentration is then moved into for 3 ‰ potassium permanganate
In solution, stirring immersion 12min；Wash liquor potassic permanganate after picking up off with clear water, then it is that 5% sodium chloride is molten to move it into concentration
Stirring immersion 10min, washes away the salt of its body surface with clear water in liquid after picking up, then with deionized water rinsing 1 time, drains moisture,
It is placed in container；
(2) by after leech body surface sterilization, calculated by leech weight, add 3 ‰ sodium hydrogensulfites, 1 ‰ ascorbic acid
Mix thoroughly, ground in input colloid mill, grinding temperature is controlled less than 50 DEG C, obtains leech slurries with 3 times of deionized waters；Described
The fineness of leech slurries is 19 mesh；
(3) the leech slurries obtained by step (2) are subjected to 2 coarse filtration, the filter residue of 2 coarse filtration of merging and filtrate；Described 2
The concrete operations of secondary coarse filtration are：1st coarse filtration is filtered with 70 mesh filter clothes in tripod pendulum type batch centrifugal, accumulates the filter residue in filter cloth with right amount
Deionized water is sprayed 2 times；2nd coarse filtration is disembarked filtering, the appropriate deionized water of filter residue accumulated in filter cloth with 200 mesh in tripodia
Spray 1 time；
(4) filtrate obtained by step (3) is subjected to micro-filtration with 30000 films that can intercept molecular weight 30000da, collects micro-filtration saturating
Cross liquid；The operating parameter of micro-filtration is：Press pump 0.2Mpa, pH value 6.5, temperature 45 C, film surface flow 2M/S, 3.5 times of cocnentration factor；
(5) micro-filtration permeate is subjected to 2 ultrafiltration, 10000 films are selected in the 1st ultrafiltration；Concentrate adds above-mentioned coarse filtration slag
In, permeate carries out the 2nd ultrafiltration, from 5000 films；It is incorporated to through solution in foregoing filter residue, gained dope is that enrichment contains
Hirudin solution；The operating parameter of the 1st time described ultrafiltration is：Press pump 0.3Mpa, pH value 5.5, temperature 45 C, crossflow velocity
4.5 times of 2M/S, cocnentration factor；The operating parameter of 2nd ultrafiltration is：Press pump 0.4Mpa, pH value are 5.5, temperature 45 C, film surface flow
6.5 times of 2M/S, cocnentration factor；
(6) coarse filtration slag and micro-filtration permeate and the dope of ultrafiltration 1, the transparent liquid of ultrafiltration 2, it is 9 to adjust pH value, and input has stirring to fill
Put, in power 5KW microwave tank, heated up with 2KW power microwaves, control temperature control is less than 60 DEG C, microwave 20min, in 58 DEG C of insulations
20min, discharge cooling；
(7) solution for discharging step (6) is cooled to 45 DEG C, is filtered, is accumulated in filter cloth with 200 mesh filter cloth tripod pendulum type batch centrifugals
Filter residue is sprayed in right amount with deionized water, and gained filtrate is leech protein solution；
(8) by leech protein solution obtained by step (7), pH value first is adjusted to 6 with watery hydrochloric acid, it is then molten with 10% glacial acetic acid again
The chitosan solution of solution 2% adjusts pH value to 5, and albumen is separated out to come, 1.5h is stood, and is used again after discarding upper liquid after solution layering
400 mesh filter cloth centrifugal filtrations, obtain wet albumen, add 2.5 times of wet liquor potassic permanganates of protein content 0.75% at a temperature of 40 DEG C
1.8h is soaked, potassium permanganate is then sloughed with 400 mesh nylon filtering cloths and is rinsed 1 time with clear water, the oxalic acid with 2.5 times of wet slags is molten
Immersion steeps 2.5h, when observation albumen is white, that is, starts to collect decolouring albumen；
(9) the wet albumen of decolouring is diluted with deionized water, when protein concentration reaches 7%, it is 9 to adjust pH value, is added
The alkaline enzyme of substrate quality 3% stirs, and is placed in band agitating device power 5KW microwave tanks, starts 2KW power, work as enzymolysis
Liquid detects enzymolysis liquid pH value when being warming up to 45 DEG C, when pH value is dropped to below 7.5, mends alkali lye and is adjusted to 9, when pH value drops to
When less than more than 7.5 8, mend alkali and be adjusted to 8.5, enzymolysis liquid is warming up to 55 DEG C and examines 1 pH value again, when pH is declined by less than less than 7.8 not
Alkali is mended again, and alkali is mended when dropping to less than 7.8 and is adjusted to 8, enzymolysis liquid temperature stops microwave when reaching 55 DEG C and retains stirring, at a temperature for the treatment of
It is reduced to after 50 DEG C and restarts 2KW power microwaves and treat that enzymolysis liquid is warming up to 55 DEG C, turns off microwave and retain stirring, treat enzymolysis liquid temperature
Enzymolysis liquid pH value is detected when dropping to less than 45 DEG C, mending alkali when pH is less than 8 is adjusted to 8, adds the pancreatin of substrate quality 2.5%,
Start 2KW microwaves, when enzymolysis liquid is warming up to 50 DEG C, check enzymolysis liquid pH value, when pH is descend below or during close to 7, mend alkali and be adjusted to
7.8, continuation microwave is warming up to 55 DEG C and turns off microwave, then detects pH, is adjusted to 7.5 when pH drops to less than 7.3 benefit alkali, startup is stirred
Mix, treat that enzymolysis liquid temperature drop restarts micropower 2KW to 45 DEG C, enzymolysis liquid is warming up to 55 DEG C, after insulation 5 minutes, start work(
Rate 10KW is warming up to 88 DEG C, is incubated 10 minutes enzyme activity of going out；The enzyme activity of described alkaline enzyme is 100,000 u/g；The enzyme activity of pancreatin>
4000u/g；
(10) enzymolysis liquid after enzyme activity of going out is discharged, is cooled to 40 DEG C, under stirring, be slowly added with containing 20% ice
The chitosan solution of acetate dissolution 1.5%, makes pH value be adjusted to 5, albumen that precipitating is not digested and other big molecular impurities etc., precipitating
Liquid stands 2 hours, and the precipitating thing being centrifuged off with 400 mesh filter clothes is leech albumen, first filters and is filtered after upper liquid during centrifugation
Dope；
(11) filtrate obtained by 400 mesh are filtered, then carry out micro-filtration with 30000 films；Dope is merged into above-mentioned 400 mesh coarse filtration
In slag；The operating parameter of micro-filtration is：Press pump 0.2Mpa, pH value 6.5, temperature 45 C, crossflow velocity 3M/S, 3.5 times of cocnentration factor；
(12) micro-filtration permeate obtained by step (11) carries out 2 ultrafiltration, and the 1st 10000 films of ultrafiltration, the 2nd ultrafiltration is used
5000 films, the concentrate of two times of ultrafiltration is merged into coarse filtration slag；The operating parameter of 1st ultrafiltration and the 2nd ultrafiltration is：Press pump
0.3Mpa, pH value 5.5, temperature 45 C, crossflow velocity 2M/S, 5.5 times of cocnentration factor；
(13) the gained permeate of ultrafiltration 2 in step (12) is subjected to sodium filter；The operating parameter of sodium filter is：Pressure
0.8Mpa, circular flow 2.0m3/ h, cycles of concentration 8.
(14) by concentrate obtained by nanofiltration in step (13), dried from spray drying process, obtain polypeptide；By step (10)
Filter residue, the dope of step (11) micro-filtration, the dope of step (12) ultrafiltration merge to be dried from spray drying process, obtains leech egg
White powder.
The present invention of table 1 produces the effect of the method for polypeptide from golden-rimmed leech
| Group | Leech Purity (%) | The enzymatic hydrolyzation (%) of leech albumen |
| Embodiment 1 | 66 | 39 |
| Embodiment 2 | 65 | 40 |
| Embodiment 3 | 68 | 43 |
As it can be seen from table 1 the present invention produces the method for polypeptide from golden-rimmed leech, to obtain leech Purity equal
More than 65%, the enzymatic hydrolyzation of leech albumen is more than 39%.
It is foregoing to the present invention specific illustrative embodiment description be in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to above-mentioned teaching, can be much changed
And change.The purpose of selecting and describing the exemplary embodiment is that explaining that the certain principles and its reality of the present invention should
With so that those skilled in the art can realize and using the present invention a variety of exemplaries and
A variety of selections and change.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (4)
1. a kind of method that polypeptide is produced from golden-rimmed leech, it is characterised in that comprise the following steps：
(1) pollutant of fresh leech or defrosting leech body surface is first rinsed with clear water, concentration is then moved into for 3 ‰ liquor potassic permanganates
In, stirring immersion 10-15min；Wash liquor potassic permanganate after picking up off with clear water, then it is that 5% sodium chloride is molten to move it into concentration
Stirring immersion 10min, washes away the salt of its body surface with clear water in liquid after picking up, then with deionized water rinsing 1 time, drains moisture,
It is placed in container；
(2) by after leech body surface sterilization, calculated by leech weight, add 3 ‰ sodium hydrogensulfites, 1 ‰ ascorbic acid and 3 times
Deionized water is mixed thoroughly, is ground in input colloid mill, grinding temperature is controlled less than 50 DEG C, obtains leech slurries；
(3) the leech slurries obtained by step (2) are subjected to 2 coarse filtration, the filter residue of 2 coarse filtration of merging and filtrate；
(4) filtrate obtained by step (3) is subjected to micro-filtration with 30000 films that can intercept molecular weight 30000Da, collects micro-filtration and pass through
Liquid；The operating parameter of micro-filtration is：Press pump 0.05-0.3Mpa, pH value 6-7,40-50 DEG C of temperature, film surface flow 1-4M/S, cocnentration factor
3-4 times；
(5) micro-filtration permeate is subjected to 2 ultrafiltration, 10000 films are selected in the 1st ultrafiltration；Concentrate is added in above-mentioned coarse filtration slag, thoroughly
Cross liquid and carry out the 2nd ultrafiltration, from 5000 films；It is incorporated to through solution in foregoing filter residue, gained dope is that enrichment contains leech
Plain solution；The operating parameter of the 1st time described ultrafiltration is：Press pump 0.1-0.43Mpa, pH value 5-6,40-50 DEG C of temperature, film surface stream
4-5 times of fast 1-3M/S, cocnentration factor；The operating parameter of 2nd ultrafiltration is：Press pump 0.3-0.6Mpa, pH value are 5-6, temperature 40-50
DEG C, film surface flow 1-3M/S, 6-7 times of cocnentration factor；
(6) coarse filtration slag and micro-filtration concentrate and the dope of ultrafiltration 1, the transparent liquid of ultrafiltration 2, it is 9 to adjust pH value, and input has agitating device,
In power 2-10KW microwave tank, heated up with 2KW power microwaves, control temperature control is less than 60 DEG C, microwave 20min, in 55-60 DEG C of insulation
20min, discharge cooling；
(7) solution for discharging step (6) is cooled to 45 DEG C, is filtered with 200 mesh filter cloth tripod pendulum type batch centrifugals, accumulates the filter residue in filter cloth
Sprayed in right amount with deionized water, gained filtrate is leech protein solution；
(8) by leech protein solution obtained by step (7), first adjust pH value to 6 with watery hydrochloric acid, then dissolved again with 10% glacial acetic acid
2% chitosan solution adjusts pH value to 5, and albumen is separated out to come, 1-2h is stood, and is used again after discarding upper liquid after solution layering
400 mesh filter cloth centrifugal filtrations, obtain wet albumen, add the 2-3 times of wet liquor potassic permanganate of protein content 0.75% in 35-45 DEG C of temperature
Lower immersion 1.5-2h, potassium permanganate is then sloughed with 400 mesh nylon filtering cloths and is rinsed 1 time with clear water, with 2-3 times of wet slag
0.75% oxalic acid solution soaks 2-3h, when observation albumen is white, that is, starts to collect decolouring albumen；
(9) the wet albumen of decolouring is diluted with deionized water, when protein concentration reaches 7%, it is 9 to adjust pH value, adds substrate
The alkaline enzyme of quality 3% stirs, and is placed in band agitating device power 2-10KW microwave tanks, starts 2KW power, work as enzymolysis liquid
Enzymolysis liquid pH value is detected when being warming up to 45 DEG C, when pH value is dropped to below more than 7.5 8, alkali is mended and is adjusted to 8.5, enzymolysis liquid heating
1 pH value is examined again to 55 DEG C, alkali is no longer mended when pH is declined by less than less than 7.8, and alkali is mended when dropping to less than 7.8 and is adjusted to 8, enzymolysis liquid
Temperature stops microwave when reaching 55 DEG C and retains stirring, restarts 2KW power microwaves after temperature drop is less than 50 DEG C and treats enzymolysis liquid
55 DEG C are warming up to, turns off microwave and retains stirring, enzymolysis liquid pH value is detected when enzymolysis liquid temperature drop is to less than 45 DEG C, when pH is low
Alkali is mended when 8 and is adjusted to 8, the pancreatin of addition substrate quality 2.5% starts 2KW microwaves, when enzymolysis liquid is warming up to 50 DEG C, checks enzyme
Liquid pH value is solved, when pH is descend below or during close to 7, alkali is mended and is adjusted to 7.8, continues microwave and be warming up to 55 DEG C to turn off microwave, then is detected
PH, is adjusted to 7.5 when pH drops to less than 7.3 benefit alkali, starts stirring, treats that enzymolysis liquid temperature drop restarts micropower to 45 DEG C
2KW, enzymolysis liquid is warming up to 55 DEG C, after being incubated 5 minutes, and startup power 10KW is warming up to 85-90 DEG C, is incubated 10 minutes enzyme activity of going out；
(10) enzymolysis liquid after enzyme activity of going out is discharged, is cooled to 40 DEG C, under stirring, be slowly added with containing 20% glacial acetic acid
The chitosan solution of dissolving 1.5%, makes pH value be adjusted to 5, albumen that precipitating is not digested and other big molecular impurities etc., and precipitating liquid is quiet
Put 2 hours, the precipitating thing being centrifuged off with 400 mesh filter clothes is leech albumen, is first filtered during centrifugation and dope is filtered after upper liquid；
(11) filtrate obtained by 400 mesh are filtered, then carry out micro-filtration with 30000 films；Dope is merged into above-mentioned 400 mesh coarse filtration slag
In；The operating parameter of micro-filtration is：Press pump 0.05-0.3Mpa, pH value 6-7,40-50 DEG C of temperature, crossflow velocity 1-4M/S, cocnentration factor
3-4 times；
(12) micro-filtration permeate carries out 2 ultrafiltration obtained by step (11), and the 1st 10000 films of ultrafiltration, the 2nd ultrafiltration uses 5000
Film, the concentrate of two times of ultrafiltration is merged into coarse filtration slag；The operating parameter of 1st ultrafiltration and the 2nd ultrafiltration is：Press pump 0.2-
0.4Mpa, pH value 5-6,40-50 DEG C of temperature, crossflow velocity 1-3M/S, 5-6 times of cocnentration factor；
(13) the gained permeate of ultrafiltration 2 in step (12) is subjected to sodium filter；The operating parameter of sodium filter is：Pressure 0.7-
0.9Mpa, circular flow 1.5-2.5m3/ h, cycles of concentration 7-9；
(14) by concentrate obtained by nanofiltration in step (13), dried from spray drying process, obtain polypeptide；By the filter of step (10)
Slag, the dope of step (11) micro-filtration, the dope of step (12) ultrafiltration merge to be dried from spray drying process, obtains leech albumen
Powder.
2. the method according to claim 1 that polypeptide is produced from golden-rimmed leech, it is characterised in that in step (2)
The fineness of gained leech slurries is 18-20 mesh.
3. the method according to claim 1 that polypeptide is produced from golden-rimmed leech, it is characterised in that in step (3)
The concrete operations of 2 times described coarse filtration are：1st coarse filtration is filtered with 60-80 mesh filter cloth in tripod pendulum type batch centrifugal, is accumulated in filter cloth
Filter residue is sprayed 1-2 times with appropriate deionized water；2nd coarse filtration is disembarked filtering with 200 mesh in tripodia, accumulates the filter residue use in filter cloth
Appropriate deionized water is sprayed 1-2 times.
4. the method according to claim 1 that polypeptide is produced from golden-rimmed leech, it is characterised in that in step (9)
The enzyme activity of described alkaline enzyme is 100,000 u/g；The enzyme activity of pancreatin>4000u/g.
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| CN107177654A (en) * | 2017-06-05 | 2017-09-19 | 深圳知本康业有限公司 | A kind of leech polypeptide powder and application |
| CN107805273A (en) * | 2017-11-16 | 2018-03-16 | 罗乌支 | A kind of method that native peptides are extracted from fly maggot |
| CN113813291B (en) * | 2021-11-10 | 2023-05-26 | 鲁南制药集团股份有限公司 | Preparation method of animal medicinal material freeze-dried powder |
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Effective date of registration: 20191226 Address after: 201, room 518000, building A, No. 1, front Bay Road, Qianhai Shenzhen Guangdong Shenzhen Hong Kong cooperation zone (Qianhai business secretary) Patentee after: Shenzhen Jhihben Kang Industry Co. Ltd. Address before: 530004 the Guangxi Zhuang Autonomous Region University of Nanning City Road No. 100 west east campus district 15 building Co-patentee before: Liang Manshui Patentee before: Liang Shangwen |