CN105906710A - Method for sequentially extracting and fractionally salting out and purifying chicken paw skin collagen protein - Google Patents
Method for sequentially extracting and fractionally salting out and purifying chicken paw skin collagen protein Download PDFInfo
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 141
- 108010035532 Collagen Proteins 0.000 title claims abstract description 141
- 241000287828 Gallus gallus Species 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000005185 salting out Methods 0.000 title abstract 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 111
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 100
- 229920001436 collagen Polymers 0.000 claims abstract description 74
- 239000011780 sodium chloride Substances 0.000 claims abstract description 49
- 239000000243 solution Substances 0.000 claims abstract description 36
- 238000003756 stirring Methods 0.000 claims abstract description 36
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000002994 raw material Substances 0.000 claims abstract description 17
- 238000001816 cooling Methods 0.000 claims abstract description 16
- 238000000605 extraction Methods 0.000 claims abstract description 9
- 239000007853 buffer solution Substances 0.000 claims abstract description 8
- 238000004108 freeze drying Methods 0.000 claims abstract description 8
- 230000007935 neutral effect Effects 0.000 claims abstract description 8
- 238000000502 dialysis Methods 0.000 claims abstract description 7
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 7
- 150000003839 salts Chemical class 0.000 claims description 44
- 239000000287 crude extract Substances 0.000 claims description 38
- 238000001556 precipitation Methods 0.000 claims description 29
- 239000000284 extract Substances 0.000 claims description 25
- 239000006228 supernatant Substances 0.000 claims description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 239000012153 distilled water Substances 0.000 claims description 21
- 239000000872 buffer Substances 0.000 claims description 20
- 102000057297 Pepsin A Human genes 0.000 claims description 16
- 108090000284 Pepsin A Proteins 0.000 claims description 16
- 239000002253 acid Substances 0.000 claims description 16
- 229940111202 pepsin Drugs 0.000 claims description 16
- 239000012043 crude product Substances 0.000 claims description 15
- 238000000746 purification Methods 0.000 claims description 14
- 238000005194 fractionation Methods 0.000 claims description 12
- 238000011010 flushing procedure Methods 0.000 claims description 9
- 239000000049 pigment Substances 0.000 claims description 9
- 235000021110 pickles Nutrition 0.000 claims description 7
- 239000008399 tap water Substances 0.000 claims description 7
- 235000020679 tap water Nutrition 0.000 claims description 7
- 239000000047 product Substances 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(I) nitrate Inorganic materials [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 4
- 238000007605 air drying Methods 0.000 claims description 3
- 102000004317 Lyases Human genes 0.000 claims description 2
- 108090000856 Lyases Proteins 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims description 2
- 230000002255 enzymatic effect Effects 0.000 claims description 2
- 210000003722 extracellular fluid Anatomy 0.000 claims description 2
- 239000000835 fiber Substances 0.000 claims description 2
- 230000002779 inactivation Effects 0.000 claims description 2
- 239000002244 precipitate Substances 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 8
- 108090000790 Enzymes Proteins 0.000 abstract description 8
- 235000013305 food Nutrition 0.000 abstract description 4
- 102000012422 Collagen Type I Human genes 0.000 abstract description 2
- 108010022452 Collagen Type I Proteins 0.000 abstract description 2
- 238000009938 salting Methods 0.000 abstract 2
- 238000004062 sedimentation Methods 0.000 abstract 2
- 238000007781 pre-processing Methods 0.000 abstract 1
- 239000012266 salt solution Substances 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 238000012545 processing Methods 0.000 description 4
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 3
- 229960002591 hydroxyproline Drugs 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 241000758706 Piperaceae Species 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000000518 rheometry Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 239000013638 trimer Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- 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
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Abstract
The invention discloses a method for sequentially extracting and fractionally salting out and purifying chicken paw skin collagen protein, and belongs to the field of food bio-utilization. The method comprises the steps that chicken paw skin with no visible fat is cleaned, dried in air under low temperature, and sheared into small blocks; the chicken paw skin is soaked into a NaCl Tris-HCl buffer solution to remove protein and fat; the pretreated chicken paw skin is soaked into a pre-cooling NaCl neutral salt solution, and stirring centrifuging is performed; sedimentations are soaked into acetic acid for extracting acid-soluble collagen, centrifuging is performed, sedimentations are taken to be soaked into an enzyme solution to extract the enzyme dissolubility collagen; the supernate obtained in the step 3 is subjected to fractional salting and dialysis, and freeze-drying is performed to obtain the pure salt-soluble collagen, the acid-soluble collagen and the enzyme-soluble collagen. According to the method, the solution is utilized for preprocessing raw materials efficiently and economically, factional extraction is utilized, the raw material utilization rate is higher, the corresponding collagen can be selected according to different application ranges, fractional salting is performed to obtain pure type-I collagen, and enzyme residues in the collagen are removed.
Description
Technical field
The invention belongs to biological food and utilize field, a kind of extraction successively and the method for salt fractionation Purified collagen.
Background technology
Broiler worldwide consumption figure is the biggest, but it is often simply discarded as the chicken feet of the by-product of poultry industry processing, for making the meat products of the simple processing such as Chicken Feet with Pickled Peppers or for producing animal feed, and a lot of western countries are never to eat chicken feet, and this has resulted in the significant wastage of resource.And containing abundant collagen protein in chicken feet skin, therefore this advantage of development and utilization chicken feet skin extracts collagen protein, can improve the economic value added of chicken feet greatly, increases its biological synthesis availability, has great meaning for Chicken industry.
I-type collagen be in body content the abundantest collagen protein, be widely present in the skin of animal, tendon and ligament.I-type collagen is widely used in medical science, food and biological field with its special structure.Chicken feet skin also contains large amounts of type i collagen protein.
The preprocess method extracting at present collagen protein from animal skins is usually, and soaks through alkali and removes pigment and foreign protein, then is removed fat by ethanol, and the method is loaded down with trivial details, and often repeatedly will clean through distilled water after step.And this method directly uses buffer solution just to reach the purpose of above several solns, it is greatly saved the time, improves efficiency.And traditional extraction process is such as: Chinese patent CN
In 105218663 A, cattle heel string collagen protein be dissolved in acetic acid pepsin solution extract, but the utilization rate of raw material is the highest, have inside substantial amounts of raw material residue possibly together with collagen protein undissolved go out.And the present invention extracts with neutral salt, acetic acid, pepsin successively, solid after each step remains next step and extracts, so can reach the effect that in raw material, collagen protein is extracted the most completely, and the feature of the collagen protein that can extract according to Different Extraction Method, use obtains local in difference, reach higher efficiency successively to extract, the purpose that more refinement collagen protein uses.And the present invention is after obtaining collagen protein crude extract, not only obtains pure I-type collagen by the method for salt fractionation, and inside the collagen protein of enzyme extraction, do not have enzyme to remain.It is convenient to extract, and raw material availability is high, and efficiency is high and obtains activated pure I-type collagen.
Summary of the invention
The by-product chicken feet skin that the present invention processes with broiler is as raw material, utilize neutral salt (NaCl), acetic acid and three kinds of solution of enzyme (Pepsin) extract, salt fractionation obtains salt soluble collagen (SSC), acid-soluble collagen protein (ASC) and the I-type collagen of pepsin-solubilized collagen (PSC) three kinds of purification successively.
Therefore, the technical solution used in the present invention is as follows: a kind of extraction successively and the method for salt fractionation purification chicken feet collagen, carries out as steps described below:
(1) pretreatment of raw material
Peel the skin on chicken feet, excise visible fat, tap water wash clean, low-temperature air-drying, be cut into small pieces.It is immersed in the Tris-HCl buffer of NaCl removing foreign protein, pigment and fat, stirring, takes centrifuged deposit.
(2) salt soluble collagen extracts
The chicken feet skin of pretreatment is soaked in the Tris-HCl buffer of the NaCl of pre-cooling, stirring, extracts, centrifuged supernatant i.e. salt soluble collagen crude extract.Centrifugation distilled water flushing.
(3) acid-soluble collagen protein extracts
Salt is centrifuged gained precipitation and is soaked in the acetic acid solution of pre-cooling after putting forward, stirring is extracted, centrifuged supernatant the most acid-soluble collagen protein crude extract.Centrifugation distilled water flushing.
(4) pepsin-solubilized collagen extracts
Acid puies forward rear gained precipitation and is soaked in the pepsin solution of pre-cooling, and stirring is extracted, centrifuged supernatant i.e. pepsin-solubilized collagen crude extract.
(5) purification of collagen protein
First adding HCl stirring in salt soluble collagen crude extract, addition NaCl is until it is completely dissolved the most while stirring, carries out saltouing overnight for the first time, centrifugal, and precipitation i.e. salt soluble collagen crude product is dissolved in acetic acid.
Directly adding NaCl in acid-soluble collagen protein crude extract, ibid step operation carries out saltouing overnight for the first time, centrifugal, and the precipitation the most acid-soluble collagen protein crude product of collection is dissolved in acetic acid.
Pepsin-solubilized collagen crude extract directly adds NaCl, ibid step operation carries out saltouing overnight for the first time, centrifugal, gained precipitated acid soluble collagen crude product, with the Tris-HCl buffer solution of NaCl, while dissolving collagen protein, neutral environment makes pepsin residual inactivation.
Collagen solution after above-mentioned three kinds of dissolvings, centrifuging and taking supernatant, again add NaCl and carry out pickle change, centrifugal collecting precipitation is dissolved in appropriate acetic acid, and distilled water is dialysed, the collagen protein dried frozen aquatic products that vacuum lyophilization must be purer.
Step (1) scalpel peels off chicken feet skin, manual excision visible fat, and chicken feet skin is cut into the square of 1cm × 1cm, and tap water rinses, and drains and air-dries at latter 20 DEG C, and-20 DEG C of preservations are to treat that next step uses.Chicken feet skin and saline solution are 0.05M than for 1g:20mL, Tris-HCl buffer, and adjusting pH is 7.5, adds 20g NaCl in 100mL buffer.It is centrifuged and carries out 20min for 10000g.This operation is repeated up to without visible fat and pigment.
In step (2), Tris-HCl buffer ibid walks the same terms, adds NaCl to 0.45mol/L in buffer, extracts 48h, and centrifugal condition is that 17000g carries out 30min.Centrifuged deposit distilled water flushing is 7.0 to pH.
In step (3), acid extracting solution is 0.5mol/L acetic acid, extracts 48h, and centrifugal condition is that 17000g carries out 30min.
In step (4), enzymatic solution is to add pepsin in 0.5mol/L acetic acid than for 0.1g:100L.Extracting 48h, centrifugal condition is that 17000g carries out 30min.
In step (5), adding HCl to 0.01mol/L in salt soluble collagen crude extract, three kinds of collagen protein crude extracts are saltoutd for the first time and are added NaCl to 0.9mol/L, and centrifugal condition is that 2500g carries out 30min for the first time.Salt carries and carries gained with acid and be precipitated and dissolved in 0.5mol/L acetic acid, and it is that 0.05mol/L adjusts pH to be 7.5 that lyase carries the Tris-HCl buffer of gained precipitation, adds NaCl to 1.0mol/L.Centrifugal condition is that 2500g carries out 30min, adds NaCl to 2.4mol/L and carry out pickle change in supernatant.After stirring, 17000g is centrifuged 30min, collects three kinds of extracting method gained precipitations, is dissolved in 0.5mol/L acetic acid, and distilled water is dialysed, until using 0.01mol/L AgNO3Till detection extracellular fluid dialysis is without white precipitate.Carry out vacuum lyophilization during collagen solution pours culture dish into afterwards, i.e. obtain the collagen protein sterling of white dried in fiber sponge shape.
The operations such as all remove impurity in above technical scheme, extract, centrifugal, purification are all carried out at 4 DEG C.
Present invention have an advantage that
(1) countries in the world consume substantial amounts of Carnis Gallus domesticus every day, but the by-product chicken feet of Chicken industry is directly considered processing fent in a lot of countries, is usually processed into feedstuff or fertilizer, and buries directly as rubbish what is more.Only part Asian countries can eat chicken feet, is the most only by simply processing, this significant wastage having resulted in resource and pollution to environment.And the present invention is with chicken feet skin as raw material, producing the collagen protein with better nutritivity value and use value, this just drastically increases its added value, increases its biological synthesis availability.
(2) tradition collagen protein need when being extracted in pretreatment of raw material carry out many more manipulations, with multiple different solution, raw material is carried out remove impurity albumen, pigment and fat, time-consumingly and will repeatedly rinse raw material with distilled water to remove previous step solution residual after wasting tank solution, and each step.And the present invention directly uses the buffer solution of neutral salt when pretreatment, can complete above troublesome operation, time saving and energy saving, efficiency is high, easily operates.
(3) containing substantial amounts of collagen protein in chicken feet skin, tradition the most only is difficult to extract the collagen protein in chicken feet skin completely by single methods such as hot water, salt, acid, thus there is raw material availability low, the problem that economic benefit collagen protein that is the highest and that extract is impure.And the present invention utilizes grading extraction method, with neutral salt, acid and enzyme, the collagen protein in chicken feet skin will be extracted the most completely successively.And can be used in foods and cosmetics field according to the textural classification of the collagen protein that three kinds of methods propose.
(4) when pepsin-solubilized collagen extracts, the thick collagen protein of purification step is dissolved in the Tris-HCl neutral buffered liquid of NaCl, purify enzyme denaturing while collagen protein, the pepsin avoided in dialysis later, lyophilizing and the step deposited in collagen protein there is also activity, it is small-molecular peptides by collagen protein enzymolysis, affects the biological activity of collagen protein.
Accompanying drawing explanation
Fig. 1: the flow chart of chicken feet collagen extracting method.
Fig. 2: different solutions extracts the SDS-PAGE collection of illustrative plates of chicken feet collagen.
The rheological behavior figure of the collagen protein that Fig. 3: different solutions extracts: the dynamic modulus (G ＇) of dynamic frequency scanning experiment.
Concrete example
Below in conjunction with example, the present invention will be described in detail:
In the present invention, three kinds of solution extract the rheology measurement of collagen protein by the following method, collagen protein is dissolved in 0.5M acetic acid preparation 1.0% (w/w) solution, with diameter 40mm grinding tool, grinding tool and sample stage spacing 1000 μm, sweep pattern, temperature 25 DEG C, frequency 0.1-10HZ, sustained stress 0.5%, carries out shaking flow measurement dynamic modulus G ＇ value.
Embodiment
1
:
To remove the chicken feet skin of visible fat, tap water wash clean, low-temperature air-drying, be cut into small pieces (about 1cm × 1cm).Weighing 100g and be immersed in Tris-HCl (0.05mol/L, the pH7.5) buffer of 20L 20% (w/v) NaCl removing foreign protein, pigment and fat, stirring 48h, 10000g are centrifuged 20min, collect precipitation, and it is 7.0 that distilled water is washed till pH.
Pretreated chicken feet skin is soaked in Tris-HCl (0.05mol/L, the pH7.5) buffer of the 0.45mol/L NaCl of 8L pre-cooling, and stirring 48h, 17000g are centrifuged 30min supernatant i.e. salt soluble collagen crude extract.Centrifuged pellet distilled water flushing is 7.0 to pH.
Salt is after carrying in the acetic acid solution of the 0.5mol/L that centrifugal gained precipitation is soaked in 8L pre-cooling, and stirring 48h, 17000g are centrifuged 30min supernatant the most acid-soluble collagen protein crude extract.
Acid puies forward rear gained precipitation and is soaked in the pepsin solution (adding 8g pepsin in 8L acetic acid) of 8L pre-cooling, and stirring 48h, 17000g are centrifuged 30min supernatant i.e. pepsin-solubilized collagen crude extract.
First adding HCl to 0.01mol/L in salt soluble collagen crude extract, add NaCl to 0.9mol/L the most while stirring and carry out saltouing overnight for the first time, 2500g is centrifuged 30min, precipitation i.e. salt soluble collagen crude product, is dissolved in 8L
In 0.5mol/L acetic acid.
Adding NaCl to 0.9mol/L in acid-soluble collagen protein crude extract the most while stirring to carry out saltouing overnight for the first time, 2500g is centrifuged 30min, the precipitation the most acid-soluble collagen protein crude product of collection, is dissolved in 8L
In 0.5mol/L acetic acid.
Pepsin-solubilized collagen crude extract adds NaCl to 0.9mol/L the most while stirring to carry out saltouing overnight for the first time, 2500g is centrifuged 30min, gained precipitated acid soluble collagen crude product, Tris-HCl (0.05mol/L, pH7.5) buffer solution with the NaCl (1.0mol/L) of 8L.
Collagen solution after above-mentioned three kinds of dissolvings, 2500g is centrifuged 30min and takes supernatant, again add NaCl to 2.4mol/L and carry out pickle change, 17000g is centrifuged 30min collection and is precipitated and dissolved in 500mL acetic acid (0.5mol/L), solution is loaded in the bag filter that molecular cut off is 14KDa, distilled water dialysis 72h, vacuum lyophilization obtains collagen protein dried frozen aquatic products.Calculating its yield according to hydroxyproline ratio in collagen protein crude extract and raw material is PSC ＞ ASC ＞ SSC, is followed successively by (49.10 ± 1.95) %, (14.49 ± 0.90) % and (1.13 ± 0.31) %.Analyzing, through SDS-PAGE, the collagen protein obtained is all purer I-type collagen.Three kinds of collagen protein dissolutions measure its rheological behavior in acetic acid, as it is shown on figure 3, three kinds of collagen solutions mainly show as elastic behavior, and mechanical loss trend is PSC ＞ ASC ＞ SSC.
Embodiment
2
:
To remove the chicken feet skin of visible fat, tap water is rinsed well, drains, and be cut into small pieces (about 1cm × 1cm).Weighing 100g and be immersed in Tris-HCl (0.05mol/L, the pH7.5) buffer of 25L 20% (w/v) NaCl removing foreign protein, pigment and fat, stirring 72h, 10000g are centrifuged 20min, collect precipitation, and it is 7.0 that distilled water is washed till pH.
Pretreated chicken feet skin is soaked in Tris-HCl (0.05mol/L, the pH7.5) buffer of the 0.45mol/L NaCl of 10L pre-cooling, and stirring 72h, 17000g are centrifuged 30min supernatant i.e. salt soluble collagen crude extract.Centrifuged pellet distilled water flushing is 7.0 to pH.
Salt is after carrying in the acetic acid solution of the 0.5mol/L that centrifugal gained precipitation is soaked in 10L pre-cooling, and stirring 72h, 17000g are centrifuged 30min supernatant the most acid-soluble collagen protein crude extract.
Acid puies forward rear gained precipitation and is soaked in the pepsin solution (adding 10g pepsin in 10L acetic acid) of 10L pre-cooling, and stirring 72h, 17000g are centrifuged 30min supernatant i.e. pepsin-solubilized collagen crude extract.
First adding HCl to 0.01mol/L in salt soluble collagen crude extract, add NaCl to 0.9mol/L the most while stirring and carry out saltouing overnight for the first time, 2500g is centrifuged 30min, precipitation i.e. salt soluble collagen crude product, is dissolved in 10L
In 0.5mol/L acetic acid.
Adding NaCl to 0.9mol/L in acid-soluble collagen protein crude extract the most while stirring to carry out saltouing overnight for the first time, 2500g is centrifuged 30min, the precipitation the most acid-soluble collagen protein crude product of collection, is dissolved in 10L
In 0.5mol/L acetic acid
Pepsin-solubilized collagen crude extract adds NaCl to 0.9mol/L the most while stirring to carry out saltouing overnight for the first time, 2500g is centrifuged 30min, gained precipitated acid soluble collagen crude product, Tris-HCl (0.05mol/L, pH7.5) buffer solution with the NaCl (1.0mol/L) of 10L.
Collagen solution after above-mentioned three kinds of dissolvings, 2500g is centrifuged 30min and takes supernatant, again add NaCl to 2.4mol/L and carry out pickle change, 17000g is centrifuged 30min collection and is precipitated and dissolved in 500mL acetic acid (0.5mol/L), distilled water dialysis 72h, vacuum lyophilization obtains collagen protein dried frozen aquatic products.Calculating its yield according to hydroxyproline ratio in collagen protein crude extract and raw material is PSC ＞ ASC ＞ SSC, is followed successively by (50.60 ± 2.21) %, (16.01 ± 0.03) % and (1.28 ± 0.97) %.When explanation is extracted compared with case 1, solid-liquid ratio uprises, yield can properly increase.Analyzing, through SDS-PAGE, the collagen protein obtained is all purer I-type collagen.
Embodiment
3
:
By removing the chicken feet skin of visible fat, tap water wash clean, draining, be cut into small pieces (about 1cm × 1cm).Weighing 100g and be immersed in Tris-HCl (0.05mol/L, the pH7.5) buffer of 15L 20% (w/v) NaCl removing foreign protein, pigment and fat, stirring 48h, 10000g are centrifuged 20min, collect precipitation, and it is 7.0 that distilled water is washed till pH.
Pretreated chicken feet skin is soaked in the 0.45mol/L of 6L pre-cooling
In Tris-HCl (0.05mol/L, the pH7.5) buffer of NaCl, stirring 48h, 17000g are centrifuged 30min supernatant i.e. salt soluble collagen crude extract.Centrifuged pellet distilled water flushing is 7.0 to pH.
Salt is after carrying in the acetic acid solution of the 0.5mol/L that centrifugal gained precipitation is soaked in 6L pre-cooling, and stirring 48h, 17000g are centrifuged 30min supernatant the most acid-soluble collagen protein crude extract.
Acid puies forward rear gained precipitation and is soaked in the pepsin solution (adding 6g pepsin in 6L acetic acid) of 6L pre-cooling, and stirring 48h, 17000g are centrifuged 30min supernatant i.e. pepsin-solubilized collagen crude extract.
First adding HCl to 0.01mol/L in salt soluble collagen crude extract, add NaCl to 0.9mol/L the most while stirring and carry out saltouing overnight for the first time, 2500g is centrifuged 30min, precipitation i.e. salt soluble collagen crude product, is dissolved in 6L
In 0.5mol/L acetic acid.
Adding NaCl to 0.9mol/L in acid-soluble collagen protein crude extract the most while stirring to carry out saltouing overnight for the first time, 2500g is centrifuged 30min, the precipitation the most acid-soluble collagen protein crude product of collection, is dissolved in 6L
In 0.5mol/L acetic acid.
Pepsin-solubilized collagen crude extract adds NaCl to 0.9mol/L the most while stirring to carry out saltouing overnight for the first time, 2500g is centrifuged 30min, gained precipitated acid soluble collagen crude product, Tris-Hcl (0.05mol/L, pH7.5) buffer solution with the NaCl (1.0mol/L) of 6L.
Collagen solution after above-mentioned three kinds of dissolvings, 2500g is centrifuged 30min and takes supernatant, again add NaCl to 2.4mol/L and carry out pickle change, 17000g is centrifuged 30min collection and is precipitated and dissolved in 500mL acetic acid (0.5mol/L), solution is loaded in the bag filter that molecular cut off is 14KDa, distilled water dialysis 72h, vacuum lyophilization obtains collagen protein dried frozen aquatic products.Calculating its yield according to hydroxyproline ratio in collagen protein crude extract and raw material is PSC ＞ ASC ＞ SSC, is followed successively by (47.78 ± 0.20) %, (13.11 ± 1.65) % and (0.78 ± 0.33) %.Analyzing, through SDS-PAGE, the collagen protein obtained is all purer I-type collagen.
The operations such as all remove impurity of above example, extract, centrifugal, purification are all carried out at 4 DEG C.The process extracting chicken feet collagen and salt fractionation successively is shown in flow chart.The collagen protein extracted can pass through its purity of SDS-PAGE map identification.The albumen that three kinds of methods obtain is all purer NTx albumen, it can be seen that SSC, ASC and PSC have three peptide chains, i.e. α clearly1And α (I)2Article (II) two, α chain, the trimer γ chain of dimer β and α of α.
Claims (6)
1. one kind is extracted and the method for salt fractionation purification chicken feet collagen successively, it is characterised in that carry out as steps described below:
(1) pretreatment of raw material
Peel the skin on chicken feet, excise visible fat, tap water wash clean, low-temperature air-drying, be cut into small pieces；It is immersed in the Tris-HCl buffer of NaCl removing foreign protein, pigment and fat, stirring, takes centrifuged deposit；
(2) salt soluble collagen extracts
The chicken feet skin of pretreatment is soaked in the Tris-HCl buffer of the NaCl of pre-cooling, stirring, extracts, centrifuged supernatant i.e. salt soluble collagen crude extract；Centrifugation distilled water flushing；
(3) acid-soluble collagen protein extracts
Salt is centrifuged gained precipitation and is soaked in the acetic acid solution of pre-cooling after putting forward, stirring is extracted, centrifuged supernatant the most acid-soluble collagen protein crude extract；Centrifugation distilled water flushing；
(4) pepsin-solubilized collagen extracts
Acid puies forward rear gained precipitation and is soaked in the pepsin solution of pre-cooling, and stirring is extracted, centrifuged supernatant i.e. pepsin-solubilized collagen crude extract；
(5) purification of collagen protein
First adding HCl stirring in salt soluble collagen crude extract, addition NaCl is until it is completely dissolved the most while stirring, carries out saltouing overnight for the first time, centrifugal, and precipitation i.e. salt soluble collagen crude product is dissolved in acetic acid；
Directly adding NaCl in acid-soluble collagen protein crude extract, ibid step operation carries out saltouing overnight for the first time, centrifugal, and the precipitation the most acid-soluble collagen protein crude product of collection is dissolved in acetic acid；
Pepsin-solubilized collagen crude extract directly adds NaCl, ibid step operation carries out saltouing overnight for the first time, centrifugal, gained precipitated acid soluble collagen crude product, with the Tris-HCl buffer solution of NaCl, while dissolving collagen protein, neutral environment makes pepsin residual inactivation；
Collagen solution after above-mentioned three kinds of dissolvings, centrifuging and taking supernatant, again add NaCl and carry out pickle change, centrifugal collecting precipitation is dissolved in appropriate acetic acid, and distilled water is dialysed, the collagen protein dried frozen aquatic products that vacuum lyophilization must be purer.
A kind of extraction successively the most according to claim 1 and the method for salt fractionation purification chicken feet collagen, it is characterized in that step (1) scalpel peels off chicken feet skin, manual excision visible fat, chicken feet skin is cut into the square of 1cm × 1cm, tap water rinses, draining and air-dry at latter 20 DEG C ,-20 DEG C of preservations are to treat that next step uses；Chicken feet skin and saline solution are 0.05M than for 1g:20mL, Tris-HCl buffer, and adjusting pH is 7.5, adds 20g NaCl in 100mL buffer；It is centrifuged and carries out 20min for 10000g；This operation is repeated up to without visible fat and pigment.
3. one kind is extracted and the method for salt fractionation purification chicken feet collagen successively, it is characterised in that in step (2), Tris-HCl buffer ibid walks the same terms, adds NaCl to 0.45mol/L in buffer, extracts 48h, and centrifugal condition is that 17000g carries out 30min；Centrifuged deposit distilled water flushing is 7.0 to pH.
4. one kind is extracted and the method for salt fractionation purification chicken feet collagen successively, it is characterised in that in step (3), acid extracting solution is 0.5mol/L acetic acid, extracts 48h, and centrifugal condition is that 17000g carries out 30min.
5. one kind is extracted and the method for salt fractionation purification chicken feet collagen successively, it is characterised in that in step (4), enzymatic solution is to add pepsin ratio in 0.5mol/L acetic acid for 0.1g:100L；Extracting 48h, centrifugal condition is that 17000g carries out 30min.
6. one kind is extracted and the method for salt fractionation purification chicken feet collagen successively, it is characterized in that in step (5), salt soluble collagen crude extract adds HCl to 0.01mol/L, three kinds of collagen protein crude extracts are saltoutd for the first time and are added NaCl to 0.9mol/L, and centrifugal condition is that 2500g carries out 30min for the first time；Salt carries and carries gained with acid and be precipitated and dissolved in 0.5mol/L acetic acid, and it is that 0.05mol/L adjusts pH to be 7.5 that lyase carries the Tris-HCl buffer of gained precipitation, adds NaCl to 1.0mol/L；Centrifugal condition is that 2500g carries out 30min, adds NaCl to 2.4mol/L and carry out pickle change in supernatant；After stirring, 17000g is centrifuged 30min, collects three kinds of extracting method gained precipitations, is dissolved in 0.5mol/L acetic acid, and distilled water is dialysed, until using 0.01mol/L AgNO3Till detection extracellular fluid dialysis is without white precipitate；Carry out vacuum lyophilization during collagen solution pours culture dish into afterwards, i.e. obtain the collagen protein sterling of white dried in fiber sponge shape.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107541538A (en) * | 2017-08-10 | 2018-01-05 | 江苏大学 | The method that in-vitro simulated gastro-intestinal digestion prepares collagen gel antioxidation polypeptide liquid |
| CN108866131A (en) * | 2018-06-11 | 2018-11-23 | 华南农业大学 | A kind of high anti-oxidation activity chicken oligopeptides and its preparation method and application |
| CN114766537A (en) * | 2022-05-24 | 2022-07-22 | 河南工业大学 | High-temperature-resistant chicken skin collagen edible casing film and preparation method thereof |
| CN117024571A (en) * | 2023-07-31 | 2023-11-10 | 中科国康(浙江)生命科学有限公司 | System and method for efficiently synthesizing recombinant humanized collagen |
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2016
- 2016-06-03 CN CN201610393886.XA patent/CN105906710A/en active Pending
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| Title |
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| LIN WANG ET AL: "Characterization of collagen from the skin of Amur sturgeon (Acipenser schrenckii)", 《FOOD HYDROCOLLOIDS》 * |
| 胡薇薇等: "鸡源胶原蛋白肽的酶法提取工艺及其自由基清除能力的研究", 《食品工业》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107541538A (en) * | 2017-08-10 | 2018-01-05 | 江苏大学 | The method that in-vitro simulated gastro-intestinal digestion prepares collagen gel antioxidation polypeptide liquid |
| CN108866131A (en) * | 2018-06-11 | 2018-11-23 | 华南农业大学 | A kind of high anti-oxidation activity chicken oligopeptides and its preparation method and application |
| CN114766537A (en) * | 2022-05-24 | 2022-07-22 | 河南工业大学 | High-temperature-resistant chicken skin collagen edible casing film and preparation method thereof |
| CN117024571A (en) * | 2023-07-31 | 2023-11-10 | 中科国康(浙江)生命科学有限公司 | System and method for efficiently synthesizing recombinant humanized collagen |
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