CN108640987A - A method of undenatured natural collagen is extracted with ionic liquid pretreatment - Google Patents
A method of undenatured natural collagen is extracted with ionic liquid pretreatment Download PDFInfo
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- CN108640987A CN108640987A CN201810666031.9A CN201810666031A CN108640987A CN 108640987 A CN108640987 A CN 108640987A CN 201810666031 A CN201810666031 A CN 201810666031A CN 108640987 A CN108640987 A CN 108640987A
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- undenatured
- natural collagen
- collagen
- ionic liquid
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- 108010035532 Collagen Proteins 0.000 title claims abstract description 155
- 102000008186 Collagen Human genes 0.000 title claims abstract description 155
- 229920001436 collagen Polymers 0.000 title claims abstract description 155
- 239000002608 ionic liquid Substances 0.000 title claims abstract description 54
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000000605 extraction Methods 0.000 claims abstract description 42
- 238000001556 precipitation Methods 0.000 claims abstract description 42
- 230000006399 behavior Effects 0.000 claims abstract description 23
- 238000012545 processing Methods 0.000 claims abstract description 15
- 239000012265 solid product Substances 0.000 claims abstract description 12
- 230000035484 reaction time Effects 0.000 claims abstract description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 90
- 239000000243 solution Substances 0.000 claims description 45
- 238000002525 ultrasonication Methods 0.000 claims description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 238000005406 washing Methods 0.000 claims description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 12
- 238000004108 freeze drying Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 238000000502 dialysis Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- -1 amine salt Chemical class 0.000 claims description 8
- 239000000284 extract Substances 0.000 claims description 7
- 239000012266 salt solution Substances 0.000 claims description 7
- IBZJNLWLRUHZIX-UHFFFAOYSA-N 1-ethyl-3-methyl-2h-imidazole Chemical compound CCN1CN(C)C=C1 IBZJNLWLRUHZIX-UHFFFAOYSA-N 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical group COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 238000009938 salting Methods 0.000 claims description 6
- 150000001412 amines Chemical class 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- SYBYTAAJFKOIEJ-UHFFFAOYSA-N 3-Methylbutan-2-one Chemical compound CC(C)C(C)=O SYBYTAAJFKOIEJ-UHFFFAOYSA-N 0.000 claims description 4
- 235000019441 ethanol Nutrition 0.000 claims description 4
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 claims description 4
- 241000270934 Rana catesbeiana Species 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 210000000664 rectum Anatomy 0.000 claims description 3
- KAIPKTYOBMEXRR-UHFFFAOYSA-N 1-butyl-3-methyl-2h-imidazole Chemical compound CCCCN1CN(C)C=C1 KAIPKTYOBMEXRR-UHFFFAOYSA-N 0.000 claims description 2
- JFYZBXKLRPWSGV-UHFFFAOYSA-N 1-methyl-3-propyl-2h-imidazole Chemical compound CCCN1CN(C)C=C1 JFYZBXKLRPWSGV-UHFFFAOYSA-N 0.000 claims description 2
- JWUJQDFVADABEY-UHFFFAOYSA-N 2-methyltetrahydrofuran Chemical compound CC1CCCO1 JWUJQDFVADABEY-UHFFFAOYSA-N 0.000 claims description 2
- NHTMVDHEPJAVLT-UHFFFAOYSA-N Isooctane Chemical compound CC(C)CC(C)(C)C NHTMVDHEPJAVLT-UHFFFAOYSA-N 0.000 claims description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 241001494479 Pecora Species 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- RAABOESOVLLHRU-UHFFFAOYSA-N diazene Chemical compound N=N RAABOESOVLLHRU-UHFFFAOYSA-N 0.000 claims description 2
- 229910000071 diazene Inorganic materials 0.000 claims description 2
- JVSWJIKNEAIKJW-UHFFFAOYSA-N dimethyl-hexane Natural products CCCCCC(C)C JVSWJIKNEAIKJW-UHFFFAOYSA-N 0.000 claims description 2
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 claims description 2
- 239000003208 petroleum Substances 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 210000002435 tendon Anatomy 0.000 claims description 2
- YDTSXOHUGRNRIA-UHFFFAOYSA-N 2-ethyl-3-methyl-1h-imidazol-3-ium;2,2,2-trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F.CCC=1NC=C[N+]=1C YDTSXOHUGRNRIA-UHFFFAOYSA-N 0.000 claims 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims 1
- ZAFNJMIOTHYJRJ-UHFFFAOYSA-N Diisopropyl ether Chemical compound CC(C)OC(C)C ZAFNJMIOTHYJRJ-UHFFFAOYSA-N 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 21
- 229940088598 enzyme Drugs 0.000 description 10
- 102000057297 Pepsin A Human genes 0.000 description 9
- 108090000284 Pepsin A Proteins 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 9
- 229940111202 pepsin Drugs 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 239000003513 alkali Substances 0.000 description 7
- 239000002994 raw material Substances 0.000 description 7
- 239000002253 acid Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 150000001450 anions Chemical class 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000010985 leather Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- 239000000515 collagen sponge Substances 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 244000309465 heifer Species 0.000 description 3
- 229920001206 natural gum Polymers 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- ZXLOSLWIGFGPIU-UHFFFAOYSA-N 1-ethyl-3-methyl-1,2-dihydroimidazol-1-ium;acetate Chemical compound CC(O)=O.CCN1CN(C)C=C1 ZXLOSLWIGFGPIU-UHFFFAOYSA-N 0.000 description 2
- SFPTVQNKTCPLAX-UHFFFAOYSA-N 3-ethyl-1-methyl-1,2-dihydroimidazol-1-ium;2,2,2-trifluoroacetate Chemical class [O-]C(=O)C(F)(F)F.CC[NH+]1CN(C)C=C1 SFPTVQNKTCPLAX-UHFFFAOYSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 108010077465 Tropocollagen Proteins 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 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
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 101000933967 Pseudomonas phage KPP25 Major capsid protein Proteins 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 239000003518 caustics Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- POLCUAVZOMRGSN-UHFFFAOYSA-N dipropyl ether Chemical compound CCCOCCC POLCUAVZOMRGSN-UHFFFAOYSA-N 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000002655 kraft paper Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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]
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of methods for extracting undenatured natural collagen with ionic liquid pretreatment, it is to be submerged to the bio-tissue for containing natural collagen in the ionic liquid that mass concentration is 50~80%, it is stirred to react 8~16h at 4~15 DEG C of temperature, after reaction time reaches, it filters out solid product and washs, be then centrifuged for processing and take precipitation；Undenatured natural collagen in precipitation obtained by centrifugal treating is extracted to get undenatured Behaviors of Collagen Solution.The present invention improves undenatured natural collagen recovery rate, and shorten extraction time by above-mentioned preprocess method.
Description
Technical field
The invention belongs to undenatured natural collagen extractive technique fields, are related to a kind of unchanged with ionic liquid pretreatment extraction
Property natural collagen method, specifically, being a kind of directly undenatured natural with the extraction of ionic liquid pretreatment bio-tissue
The method of collagen.
Background technology
Collagen (collagen) is a kind of naturally occurring celloglobulin, and molecular weight is 300,000, and structure is still protected
Hold the triple helix structure of collagen, have bioactivity, be primarily present in the tissues such as the skin, bone, tendon of animal, be human body and
The major structural protein matter of vertebrate plays a part of supporting organ, protection body.Collagen is that in-vivo content is most
Protein accounts for the 25%~30% of vivo protein total amount, is equivalent to the 6% of weight, and skin contains 50% or more collagen.It is undenatured
Natural collagen is a kind of fibrin extracted from the tissues such as the skin, heel string and cartilage of animal, and relative molecular mass is about 3
×105, it holds triple helix structures, thus have bioactivity.
The extracting method of collagen mainly has acid system, alkaline process, acid-enzyme binding-method and alkali enzyme combined techniques.Traditional extraction
Method, as acid-enzyme binding-method collagen yield is generally below 20%, (Zhang Wenxiong, Li Xin wait《The research of Collagen Extraction with Enzyme》,
Chinese leather, 2006,35 (23)：15-17), and extraction time is longer.Therefore it researchs and proposes by optimizing pretreatment to carry
High collagen yield, skin is pre-processed for example, by using two-step method (it is pay strong,《Natural collagen improves the skin pretreatment of recovery rate
The research of method》, Chinese leather, 2006,35 (1)：28-35), alkali effect, enzymatic treatment, then two steps with hydrogen peroxide are first passed through
Preprocess method removes most of interfibrillar substance, by pretreatment although can within a short period of time eliminate the hair on skin
The recovery rate of optimization also only reaches 30%, and extraction time is also generally in 48~72h.
With the continuous development of natural collagen related application, demand is continuously increased, accordingly in undenatured natural collagen
Recovery rate is improved in preparation process, reduction extraction time has become inevitable development trend.
Invention content
In view of the deficiencies of the prior art, it is an object of the present invention to provide one kind extracting undenatured day with ionic liquid pretreatment
The method of right collagen, improves undenatured natural collagen recovery rate, and shorten extraction time.
To achieve the above object, the present invention is realized using the technical solution being made of following technical measures.
A method of undenatured natural collagen is extracted with ionic liquid pretreatment, is the organism group that will contain natural collagen
It knits and is submerged in the ionic liquid that mass concentration is 50~80%, 8~16h, reaction time are stirred to react at 4~15 DEG C of temperature
It after arrival, filters out solid product and washs, be then centrifuged for processing and take precipitation；
Undenatured natural collagen in precipitation obtained by centrifugal treating is extracted to get undenatured Behaviors of Collagen Solution；
The ionic liquid includes 1- ethyl-3-methylimidazole dintrile amine salt solution, 1- butyl -3- methylimidazole dintrile amine
Salting liquid, -3 methylimidazole villaumite solution of 1- octyls, 1- ethyl-3-methylimidazoles Acetate Solution, 1- ethyl-3-methylimidazoles
Tetrafluoro boric acid salting liquid, 1- ethyl-3-methylimidazoles phosphate dihydrogen salt solution, 1- propyl -3- methylimidazole diimine salting liquids
With any one in 1- ethyl-3-methylimidazole trifluoroacetates.
Normally, the bio-tissue containing natural collagen and the respective additive amount of ionic liquid disclosure satisfy that by containing
The bio-tissue of natural collagen is not limited in the case of being totally submerged to ionic liquid, but in order to better illustrate this
Invention usually selects suitable reaction vessel, then extremely with weight ratio according to the bio-tissue additive amount containing natural collagen
It is 1 less：10 mode adds bio-tissue and ionic liquid containing natural collagen.
Wherein, the bio-tissue containing natural collagen is the bio-tissue rich in natural collagen, such as ox-hide, pig
Any one of skin, fish-skin, sheepskin, bullfrog skin, snakeskin, ox heel string, pig heel string and sheep heel string.
Normally, described containing natural in order to preferably utilize bio-tissue of the ionic liquid pretreatment containing natural collagen
The bio-tissue of collagen is the bulk for being not more than 5mm for latus rectum by peace and quiet and processing before immersing ionic liquid.It is excellent
It is bulk that latus rectum is 0.5~1mm that selection of land, which is by the bio-tissue shear treatment for containing natural collagen,.
Wherein, described to filter out solid product and wash, to filter out solid product and being washed with deionized 4~6 times.
Further, described to filter out solid product and be washed with deionized 4~6 times, further include in washing process
With ultrasonication.
Further, the ultrasonication is the ultrasonic equipment for using power as 50~100w, and at every ultrasonic wave
10~20min is managed, 5~10min of ultrasonication is just stopped, being repeated 3~5 times.
Wherein, the centrifugal treating is that 8000~15000rpm of rotating speed is centrifuged until supernatant is clarified, and supernatant is gone to take
Precipitation.
Normally, the centrifugal treating takes precipitation, further include by precipitation obtained by centrifugal treating further use organic solvent into
Row washing, the organic solvent includes acetone, ethyl alcohol, polyethylene glycol, ether, methyl phenyl ethers anisole, 1- propyl alcohol, 2- propyl alcohol, isooctane, different
Any one of propyl ether, methyl isopropyl ketone, dimethyl sulfoxide (DMSO), methyltetrahydrofuran and petroleum ether.
Wherein, described to filter out solid product and wash, further include recycling filtrate, then revolving is made after removing distilled water
It is reused for ionic liquid.
Wherein, described to extract undenatured natural collagen in precipitation obtained by centrifugal treating, being will be obtained by centrifugal treating
Precipitation is taken using undenatured natural gum ortho acid enzyme extraction method, undenatured natural collagen acid extraction method, undenatured natural collagen alkali carries
Method or undenatured natural collagen alkali enzyme extraction method extract undenatured natural collagen.It is noted that above-mentioned undenatured natural
Collagenic acid enzyme extraction method, undenatured natural collagen acid extraction method, undenatured natural collagen alkali extraction method or undenatured natural collagen
Alkali enzyme extraction method, to adopt by reference existing undenatured natural collagen extraction method, (Xiao Shiwei, Zeng Rui wait《It is carried from Animal Skin
The method for taking collagen》, western leather, 2009,31 (15)：17-21；Wang Lu waits《The preparation of type i collagen and performance table
Sign》, biomedical engineering and clinical, 2018,22 (1)：104-109).
In order to further improve undenatured natural collagen recovery rate, and shorten extraction time, it is described to centrifugal treating institute
Undenatured natural collagen in must precipitating extracts, and includes the following steps：
Count in parts by weight, by precipitation obtained by 10 parts of centrifugal treatings be added to 350~400 parts of molar concentrations be 0.4~
In 0.6mol/L acetic acid and 0.25~0.35 part of pepsin, it is stirred to react 12~16h of extraction, it is molten to obtain undenatured natural collagen
Liquid.
Further, in order to reduce the impurity in the undenatured Behaviors of Collagen Solution of gained, extraction gained is undenatured natural
The purified processing of collagen solution, the purification process is conventional undenatured natural collagen purification treating method, in order to preferably say
The bright present invention, it is preferable that the purification process is that will extract acquired solution centrifugation, takes supernatant that molar concentration is added and is
The NaCl solution of 0.5~3mol/L is saltoutd, and the gained precipitation that will saltout, which is centrifuged, takes centrifuged deposit molar concentration
For the acetic acid of 0.05~1mol/L, aforesaid operations are repeated 2~4 times, then molten with the acetic acid that molar concentration is 0.1~1mol/L
Liquid is dialyzate dialysis treatment 18~for 24 hours.
Further, it preserves and transports for convenience, usually may be selected the undenatured natural gum after above-mentioned purification process
Original solution is lyophilized, and obtains spongiform undenatured natural collagen.
The present invention is using the highly polar of ionic liquid, and the zwitterion in ionic liquid to being dissociated into free sun first
Ion and anion, the H on then in these free anion and collagen peptide bond-NH-coordinates, and cation and collagen
Peptide bond C=O is combined.Simultaneously because the effect of electrostatic force, cation and anion with opposite charges respectively with collagen side chain
The COO of ionization-With NH3 +Attract each other bonding, destroys COO-With NH3 +Between original electrovalent bond.Cation and anion and
The hydrogen bond and ionic bond newly formed between tropocollagen molecule has interrupted hydrogen bond and ionic bond between tropocollagen molecule.Because it is interfibrous away from
From increase, collagenous fibres have been properly dispersed.And materials with hide glue fibril disperses better, protease more holds when natural collagen extracts
Easily and substrate-function, to which natural collagen meltage is more, recovery rate is higher, and the time needed is shorter.
Wherein, it is pointed out that collagenous fibres need to need to accomplish suitable degree of reaction, reaction interval in ionic liquid
High easy excess destruction collagen structure is spent, and collagenolysis is made to be difficult to extract in ionic liquid；On the contrary, reaction interval is spent
It is low, it is easy so that underaction causes evacuation degree inadequate, recovery rate reduces.And reaction interval of the collagenous fibres in ionic liquid
Proportional relation is presented in the concentration of degree and ionic liquid, and ionic liquid concentration is too high to be easy to cause too strong destroy collagen three strands of reaction
Helical structure, ionic liquid concentration is too low and fiber evacuation degree can be caused inadequate.Therefore present invention defines ionic liquid constitutions
Measure a concentration of 50~80% so that ionic liquid concentration is moderate, to ensure high extraction and short extraction time.
The present invention has the advantages that：
1, the invention discloses the ionic liquid pretreatment bio-tissue using moderate concentration, loose collagenous fibres knots
The recovery rate of structure, collagen is higher than 75%.
2, the present invention is because pre-processing the bio-tissue containing natural collagen using ionic liquid, to undenatured natural
Substantially shorten the time required to collagen extraction process, in the case where recovery rate is higher than 75%, to after pretreatment containing natural collagen
It can be less than 16h the time required to bio-tissue extraction collagen.
3, the present invention extracts undenatured natural collagen and remains the distinctive triple helix structure of undenatured natural collagen, protects
The bioactivity of collagen is demonstrate,proved, gained collagen has low immunogenicity, biocompatibility and biodegradability.
4, ionic liquid used in present invention pretreatment can be recycled and be reused.
Description of the drawings
Fig. 1 is the gel electrophoresis figure of the embodiment of the present invention 1~5.In figure, Maker is Protein standards, 1,2,3,4,5
The undenatured natural collagen of the extraction of corresponding embodiment 1~5 gained respectively is to use sodium dodecyl sulfate-polypropylene acrylamide gel
Electrophoresis (SDS-PAGE) carries out molecular weight determination to the undenatured natural collagen that Examples 1 to 5 is extracted, and gained natural gum original sample is all
With apparent α components and β components, wherein α components are made of 1 peptide chains of adjacent α and 2 peptide chains of α, and have no other bands,
Illustrate that its molecular mass is concentrated, to maintain the natural collagen of triple helix structure feature, and purity is higher.
Fig. 2 is the sectional view of 1 heifer of embodiment of the present invention ox-hide histotomy after impregnating reaction by ionic liquid.
Fig. 3 is the longitudinal sectional figure of 1 heifer of embodiment of the present invention ox-hide histotomy after impregnating reaction by ionic liquid.
Fig. 4 is the sectional view without pretreated ox-hide histotomy in comparative example 1 of the present invention.
Fig. 5 is the longitudinal sectional figure without pretreated ox-hide histotomy in comparative example 1 of the present invention.
Specific implementation mode
The invention will be further described by way of example and in conjunction with the accompanying drawings.It is worth noting that the implementation provided
Example should not be understood as limiting the scope of the invention, and person skilled in art's content according to the present invention makees the present invention
Some the nonessential modifications and adaptations gone out should belong to the scope of the present invention.
Collagen recovery rate calculates
By the hydroxyproline content of the collagen sponge after bio-tissue of the measurement containing natural collagen and freeze-drying, to determine
Collagen content in collagen sponge after bio-tissue and freeze-drying containing natural collagen, to calculate collagen yield w (%), such as
Following formula：
Wherein, collagen quality (g) in sponge obtained by m1-experiment；
Collagen quality (g) in m2-bio-tissue.
Embodiment 1
(1) pretreatment of raw material
It counts in parts by weight, 10 parts of ox-hides are peace and quiet, be cut into length, width and height 0.5mm fragments, being submerged into 100 parts of mass concentrations is
In 70% 1- ethyl-3-methylimidazole dintrile amine salt solutions, it is stirred to react 12h, solid production is filtered out after reaction time arrival
Object is simultaneously washed with deionized, and carries out ultrasonication with power for 50W ultrasonic equipments in washing process, and per ultrasound
Wave handles 10min, just stops ultrasonication 5min, is repeated 3 times, and is then centrifuged for processing and takes precipitation, after washing centrifugation with acetone
Precipitation, removes remaining ionic liquid.
(2) extraction of undenatured natural collagen
It counts in parts by weight, by 10 parts of step (1) centrifugal treatings and washs gained precipitation and be added to 400 parts of molar concentrations and be
In 0.5mol/L acetic acid and 0.3 part of pepsin, it is stirred to react extraction 16h, obtains undenatured Behaviors of Collagen Solution.
(3) purifying of undenatured natural collagen
Undenatured Behaviors of Collagen Solution obtained by step (2) is centrifuged with supercentrifuge, filter residue is removed, takes supernatant
The middle molar concentration that is added is saltoutd for the NaCl of 0.7mol/L, and the gained precipitation that will saltout, which is centrifuged, takes centrifuged deposit to use
Molar concentration is the acetic acid of 0.5mol/L, repeats aforesaid operations 2 times, then with molar concentration is the acetic acid solution of 0.5mol/L
For dialyzate dialysis treatment 22h, freeze-drying obtains spongiform undenatured natural collagen.
In above step unless otherwise specified, it is carried out at 4~15 DEG C of temperature.
It is computed, the recovery rate of final undenatured natural collagen is 85.2%.
Attached drawing 2,3 be respectively the present embodiment heifer by ionic liquid impregnate reaction after ox-hide histotomy it is crosscutting
Figure and longitudinal sectional figure, it can be seen that kraft fibers spacing significantly increases, and fiber has obtained effective loose.
Embodiment 2
(1) pretreatment of raw material
It counts in parts by weight, 10 parts of pigskins are peace and quiet, be cut into length, width and height 0.6mm fragments, being submerged into 100 parts of mass concentrations is
In 50% 1- ethyl-3-methylimidazole Acetate Solutions, it is stirred to react 9h, solid product is filtered out simultaneously after reaction time arrival
It is washed with deionized, carries out ultrasonication with power for 60W ultrasonic equipments in washing process, and at every ultrasonic wave
11min is managed, just stops ultrasonication 6min, is repeated 4 times, processing is then centrifuged for and takes precipitation, centrifuged deposit is washed with ethyl alcohol,
Remove remaining ionic liquid.
(2) extraction of undenatured natural collagen
It counts in parts by weight, by 10 parts of step (1) centrifugal treatings and washs gained precipitation and be added to 400 parts of molar concentrations and be
In 0.6mol/L acetic acid and 0.3 part of pepsin, it is stirred to react extraction 15h, obtains undenatured Behaviors of Collagen Solution.
(3) purifying of undenatured natural collagen
Undenatured Behaviors of Collagen Solution obtained by step (2) is centrifuged with supercentrifuge, filter residue is removed, takes supernatant
The middle molar concentration that is added is saltoutd for the NaCl of 0.8mol/L, and the gained precipitation that will saltout, which is centrifuged, takes centrifuged deposit to use
Molar concentration is the acetic acid of 0.6mol/L, repeats aforesaid operations 2 times, then with molar concentration is the acetic acid solution of 0.6mol/L
For dialyzate dialysis treatment 20h, freeze-drying obtains spongiform undenatured natural collagen.
In above step unless otherwise specified, it is carried out at 4~15 DEG C of temperature.
It is computed, the recovery rate of final undenatured natural collagen is 82.6%.
Embodiment 3
(1) pretreatment of raw material
It counts in parts by weight, it is 10 parts of ox heel strings are peace and quiet, be cut into length, width and height 0.6mm fragments, it is submerged into 100 parts of mass concentrations
In 80% 1- ethyl-3-methylimidazole tetrafluoro boric acid salting liquids, to be stirred to react 13h, filtered out after reaction time arrival solid
Body product is simultaneously washed with deionized, and carries out ultrasonication with power for 65W ultrasonic equipments in washing process, and every
Ultrasonication 12min just stops ultrasonication 7min, is repeated 5 times, and is then centrifuged for processing and takes precipitation, uses dimethyl sulfoxide (DMSO)
Centrifuged deposit is washed, remaining ionic liquid is removed.
(2) extraction of undenatured natural collagen
It counts in parts by weight, by 10 parts of step (1) centrifugal treatings and washs gained precipitation and be added to 400 parts of molar concentrations and be
In 0.4mol/L acetic acid and 0.3 part of pepsin, it is stirred to react extraction 14h, obtains undenatured Behaviors of Collagen Solution.
(3) purifying of undenatured natural collagen
Undenatured Behaviors of Collagen Solution obtained by step (2) is centrifuged with supercentrifuge, filter residue is removed, takes supernatant
The middle molar concentration that is added is saltoutd for the NaCl of 0.6mol/L, and the gained precipitation that will saltout, which is centrifuged, takes centrifuged deposit to use
Molar concentration is the acetic acid of 0.4mol/L, repeats aforesaid operations 2 times, then with molar concentration is the acetic acid solution of 0.4mol/L
For dialyzate dialysis treatment 18h, freeze-drying obtains spongiform undenatured natural collagen.
In above step unless otherwise specified, it is carried out at 4~15 DEG C of temperature.
It is computed, the recovery rate of final undenatured natural collagen is 75.4%.
Embodiment 4
(1) pretreatment of raw material
It counts in parts by weight, 10 parts of fish-skins are peace and quiet, be cut into length, width and height 0.5mm fragments, being submerged into 100 parts of mass concentrations is
In 65% 1- butyl -3- methylimidazole phosphate dihydrogen salt solutions, it is stirred to react 10h, solid is filtered out after reaction time arrival
Product is simultaneously washed with deionized, and carries out ultrasonication with power for 50W ultrasonic equipments in washing process, and often surpass
Sonicated 12min, just stop ultrasonication 8min, be repeated 4 times, be then centrifuged for processing take precipitation, with 1- acetone wash from
It is precipitated after the heart, removes remaining ionic liquid.
(2) extraction of undenatured natural collagen
It counts in parts by weight, by 10 parts of step (1) centrifugal treatings and washs gained precipitation and be added to 400 parts of molar concentrations and be
In 0.55mol/L acetic acid and 0.3 part of pepsin, it is stirred to react extraction 14h, obtains undenatured Behaviors of Collagen Solution.
(3) purifying of undenatured natural collagen
Undenatured Behaviors of Collagen Solution obtained by step (2) is centrifuged with supercentrifuge, filter residue is removed, takes supernatant
The middle molar concentration that is added is saltoutd for the NaCl of 0.75mol/L, and the gained precipitation that will saltout, which is centrifuged, takes centrifuged deposit
The acetic acid for being 0.55mol/L with molar concentration repeats aforesaid operations 2 times, then with molar concentration is the acetic acid of 0.55mol/L
For 24 hours for dialyzate dialysis treatment, freeze-drying obtains spongiform undenatured natural collagen to solution.
In above step unless otherwise specified, it is carried out at 4~15 DEG C of temperature.
It is computed, the recovery rate of final undenatured natural collagen is 79.8%.
Embodiment 5
(1) pretreatment of raw material
It counts in parts by weight, it is 10 parts of bullfrog skins are peace and quiet, be cut into length, width and height 0.7mm fragments, it is submerged into 100 parts of mass concentrations
In 71% 1- ethyl-3-methylimidazole trifluoroacetic acid salting liquids, to be stirred to react 13h, filtered out after reaction time arrival solid
Body product is simultaneously washed with deionized, and carries out ultrasonication with power for 85W ultrasonic equipments in washing process, and every
Ultrasonication 10min, just stop ultrasonication 7min, be repeated 4 times, be then centrifuged for processing take precipitation, with ether wash from
It is precipitated after the heart, removes remaining ionic liquid.
(2) extraction of undenatured natural collagen
It counts in parts by weight, by 10 parts of step (1) centrifugal treatings and washs gained precipitation and be added to 350 parts of molar concentrations and be
In 0.45mol/L acetic acid and 0.26 part of pepsin, it is stirred to react extraction 12h, obtains undenatured Behaviors of Collagen Solution.
(3) purifying of undenatured natural collagen
Undenatured Behaviors of Collagen Solution obtained by step (2) is centrifuged with supercentrifuge, filter residue is removed, takes supernatant
The middle molar concentration that is added is saltoutd for the NaCl of 0.73mol/L, and the gained precipitation that will saltout, which is centrifuged, takes centrifuged deposit
The acetic acid for being 0.45mol/L with molar concentration repeats aforesaid operations 3 times, then with molar concentration is the acetic acid of 0.45mol/L
Solution is dialyzate dialysis treatment 18h, and freeze-drying obtains spongiform undenatured natural collagen.
In above step unless otherwise specified, it is carried out at 4~15 DEG C of temperature.
It is computed, the recovery rate of final undenatured natural collagen is 76.3%.
Comparative example 1
(1) extraction of undenatured natural collagen
It counts in parts by weight, 10 parts of ox-hides are peace and quiet, be cut into length, width and height 0.5mm fragments, being added to 400 parts of molar concentrations is
In 0.5mol/L acetic acid and 0.3 part of pepsin, it is stirred to react extraction 16h, obtains undenatured Behaviors of Collagen Solution.
(2) purifying of undenatured natural collagen
Undenatured Behaviors of Collagen Solution obtained by step (1) is centrifuged with supercentrifuge, filter residue is removed, takes supernatant
The middle molar concentration that is added is saltoutd for the NaCl of 0.7mol/L, and the gained precipitation that will saltout, which is centrifuged, takes centrifuged deposit to use
Molar concentration is the acetic acid of 0.5mol/L, repeats aforesaid operations 2 times, then with molar concentration is the acetic acid solution of 0.5mol/L
For dialyzate dialysis treatment 22h, freeze-drying obtains spongiform undenatured natural collagen.
It is computed, the recovery rate of final undenatured natural collagen is 19%.It can easily be seen that comparative example 1, without pre-
The collagen yield of the ox-hide of processing is far below 1 effect of the embodiment of the present invention.
Comparative example 2
Reference literature (it pays by force,《Natural collagen improves the research of the skin preprocess method of recovery rate》, Chinese leather, 2006,
35(1)：Preprocess method in 28-35) applies enzyme paste in the grain of skin and acts on certain time, by machine by ox-hide after caustic dip
Tool power removes hair, then the skin that alkali, enzyme effect are crossed, and 4.5h is handled under conditions of pH is 10 with 60g/L hydrogen peroxide.Then
Pretreated skin is shredded, at moderate temperatures with suitable Protease Treatment, gained collagen is through centrifuging, saltouing, dialyse
Process finally makes collagen sponge using freeze-drying.
It is computed, the recovery rate of final undenatured natural collagen is 30%.
Comparative example 3
(1) pretreatment of raw material
It counts in parts by weight, 10 parts of pigskins are peace and quiet, be cut into length, width and height 0.6mm fragments, being submerged into 100 parts of mass concentrations is
In 90% 1- ethyl-3-methylimidazole Acetate Solutions, it is stirred to react 9h, solid product is filtered out simultaneously after reaction time arrival
It is washed with deionized, carries out ultrasonication with power for 60W ultrasonic equipments in washing process, and at every ultrasonic wave
11min is managed, just stops ultrasonication 6min, is repeated 4 times, processing is then centrifuged for and takes precipitation, centrifuged deposit is washed with ethyl alcohol,
Remove remaining ionic liquid.
(2) extraction of undenatured natural collagen
It counts in parts by weight, by 10 parts of step (1) centrifugal treatings and washs gained precipitation and be added to 400 parts of molar concentrations and be
In 0.6mol/L acetic acid and 0.3 part of pepsin, it is stirred to react extraction 15h, obtains undenatured Behaviors of Collagen Solution.
(3) purifying of undenatured natural collagen
Undenatured Behaviors of Collagen Solution obtained by step (2) is centrifuged with supercentrifuge, filter residue is removed, takes supernatant
The middle molar concentration that is added is saltoutd for the NaCl of 0.8mol/L, and the gained precipitation that will saltout, which is centrifuged, takes centrifuged deposit to use
Molar concentration is the acetic acid of 0.6mol/L, repeats aforesaid operations 2 times, then with molar concentration is the acetic acid solution of 0.6mol/L
For dialyzate dialysis treatment 20h, freeze-drying obtains spongiform undenatured natural collagen.
In above step unless otherwise specified, it is carried out at 4~15 DEG C of temperature.
It is computed, the recovery rate of final undenatured natural collagen is 48.7%.It can easily be seen that comparative example 2, pretreatment
The excessively high decline for instead resulting in collagen yield of ionic liquid concentration in the process.
Comparative example 4
(1) pretreatment of raw material
It counts in parts by weight, 10 parts of ox-hides are peace and quiet, be cut into length, width and height 0.5mm fragments, being submerged into 100 parts of mass concentrations is
In 20% 1- ethyl-3-methylimidazole dintrile amine salt solutions, it is stirred to react 12h, solid production is filtered out after reaction time arrival
Object is simultaneously washed with deionized, and carries out ultrasonication with power for 50W ultrasonic equipments in washing process, and per ultrasound
Wave handles 10min, just stops ultrasonication 5min, is repeated 3 times, and is then centrifuged for processing and takes precipitation, after washing centrifugation with acetone
Precipitation, removes remaining ionic liquid.
(2) extraction of undenatured natural collagen
It counts in parts by weight, by 10 parts of step (1) centrifugal treatings and washs gained precipitation and be added to 400 parts of molar concentrations and be
In 0.5mol/L acetic acid and 0.3 part of pepsin, it is stirred to react extraction 16h, obtains undenatured Behaviors of Collagen Solution.
(3) purifying of undenatured natural collagen
Undenatured Behaviors of Collagen Solution obtained by step (2) is centrifuged with supercentrifuge, filter residue is removed, takes supernatant
The middle molar concentration that is added is saltoutd for the NaCl of 0.7mol/L, and the gained precipitation that will saltout, which is centrifuged, takes centrifuged deposit to use
Molar concentration is the acetic acid of 0.5mol/L, repeats aforesaid operations 2 times, then with molar concentration is the acetic acid solution of 0.5mol/L
For dialyzate dialysis treatment 22h, freeze-drying obtains spongiform undenatured natural collagen.
In above step unless otherwise specified, it is carried out at 4~15 DEG C of temperature.
It is computed, the recovery rate of final undenatured natural collagen is 51.2%.It can easily be seen that comparative example 1, pretreatment
Ionic liquid concentration is too low so that collagen yield declines in the process.
Claims (10)
1. a kind of method for extracting undenatured natural collagen with ionic liquid pretreatment, it is characterised in that natural collagen will be contained
Bio-tissue is submerged in the ionic liquid that mass concentration is 50~80%, and 8~16h is stirred to react at 4~15 DEG C of temperature,
It after reaction time reaches, filters out solid product and washs, be then centrifuged for processing and take precipitation；
Undenatured natural collagen in precipitation obtained by centrifugal treating is extracted to get undenatured Behaviors of Collagen Solution；
The ionic liquid includes that 1- ethyl-3-methylimidazole dintrile amine salt solution, 1- butyl -3- methylimidazole dintrile amine salt are molten
Liquid, -3 methylimidazole villaumite solution of 1- octyls, 1- ethyl-3-methylimidazoles Acetate Solution, 1- ethyl-3-methylimidazole tetrafluoros
Borate solution, 1- ethyl-3-methylimidazoles phosphate dihydrogen salt solution, 1- propyl -3- methylimidazole diimine salting liquids and 1-
Any one in ethyl-3-methylimidazole trifluoroacetate.
2. the method for extracting undenatured natural collagen according to claim 1 with ionic liquid pretreatment, it is characterised in that institute
State the bio-tissue containing natural collagen be ox-hide, pigskin, fish-skin, sheepskin, bullfrog skin, snakeskin, ox heel string, pig heel string and sheep with
Any one of tendon.
3. the method according to claim 1 or claim 2 for extracting undenatured natural collagen with ionic liquid pretreatment, it is characterised in that
The bio-tissue containing natural collagen is to be not more than 5mm by peace and quiet and processing for latus rectum before immersing ionic liquid
Bulk.
4. the method for extracting undenatured natural collagen according to claim 1 with ionic liquid pretreatment, it is characterised in that institute
It states and filters out solid product and wash, to filter out solid product and being washed with deionized 4~6 times.
5. the method for extracting undenatured natural collagen according to claim 4 with ionic liquid pretreatment, it is characterised in that institute
It states and filters out solid product and be washed with deionized 4~6 times, further include the adjoint ultrasonication in washing process.
6. the method for extracting undenatured natural collagen according to claim 5 with ionic liquid pretreatment, it is characterised in that institute
It is the ultrasonic equipment for using power as 50~100w to state ultrasonication, and per 10~20min of ultrasonication, is just stopped super
5~10min of sonicated is repeated 3~5 times.
7. the method for extracting undenatured natural collagen according to claim 1 with ionic liquid pretreatment, it is characterised in that institute
It states centrifugal treating and takes precipitation, further include further washing precipitation obtained by centrifugal treating with organic solvent；It is described organic molten
Agent include acetone, ethyl alcohol, polyethylene glycol, ether, methyl phenyl ethers anisole, 1- propyl alcohol, 2- propyl alcohol, isooctane, isopropyl ether, methyl isopropyl ketone,
Any one of dimethyl sulfoxide (DMSO), methyltetrahydrofuran and petroleum ether.
8. the method for extracting undenatured natural collagen according to claim 1 with ionic liquid pretreatment, it is characterised in that institute
It states and the undenatured natural collagen in precipitation obtained by centrifugal treating is extracted, include the following steps：
It counts in parts by weight, it is 0.4-0.6mol/L that precipitation obtained by 10 parts of centrifugal treatings, which is added to 350-400 parts of molar concentrations,
In acetic acid and 0.25-0.35 parts of pepsins, it is stirred to react extraction 12-16h, obtains undenatured Behaviors of Collagen Solution.
9. the method for extracting undenatured natural collagen according to claim 1 with ionic liquid pretreatment, it is characterised in that also
Including that will extract the undenatured purified processing of Behaviors of Collagen Solution of gained, the purification process is that will extract acquired solution centrifugation point
From, take supernatant be added molar concentration be 0.5~3mol/L NaCl solution saltout, will saltout gained precipitation through centrifugation point
From taking centrifuged deposit molar concentration to be the acetic acid of 0.05~1mol/L, repeat aforesaid operations 2~4 times, then with mole
The acetic acid solution of a concentration of 0.1~1mol/L is dialyzate dialysis treatment 18~for 24 hours.
10. the method for extracting undenatured natural collagen according to claim 9 with ionic liquid pretreatment, it is characterised in that will
Undenatured Behaviors of Collagen Solution freeze-drying after the purification process, obtains spongiform undenatured natural collagen.
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