CN101182358A - Purification method for preparing antler collagen - Google Patents
Purification method for preparing antler collagen Download PDFInfo
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- CN101182358A CN101182358A CNA2007101935410A CN200710193541A CN101182358A CN 101182358 A CN101182358 A CN 101182358A CN A2007101935410 A CNA2007101935410 A CN A2007101935410A CN 200710193541 A CN200710193541 A CN 200710193541A CN 101182358 A CN101182358 A CN 101182358A
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Abstract
The invention relates to a method of separating and extracting antler collagen. The concrete steps are that the antler is extracted by Tris-HCl buffer solution homogenate solution for centrifugation and deposition to be washed cleanly by the Tris-HCl buffer solution, 70 percent of ethanol is added to extract under 4 DEG C for48 hours and then for reflux extraction under 60 DEG C, the ethanol is recycled by rotary evaporation after 48 hours, extract liquid is put into a dialyzing bag with the cutoff molecular weight of 8 to 15kDa, distilled water is dialyzed for 24 hours, pre-frozen under subzero 20 DEG C for 24 hours and frozen to be dry, and the antler collagen with the content of about 70 percent is obtained. Compared with the prior collagen extraction method, the antler collagen extraction technology of the invention is simple and convenient and is easy to apply; activity experiment shows that the antler collagen has the effect of promoting the cell growth and still keeps the biological activity, and our extraction method does not damage the natural conformation of the collagen.
Description
Technical field
The invention belongs to biological product technical field, be specifically related to a kind of adopt in turn neutral, acidic buffer, combining by technology such as ethanol lixiviate, refluxing extraction prepares the separation purification method of antler collagen.
Background technology
Before the sixties in 20th century, often protein is referred to as protein, therefore collagen is referred to as the glue protein, mean the protein of colloidality.The protein that given up the sixties is called protein, and the title of collagen has also just been arranged.Someone is called hydrolytic collagen or gelatin hydrolysate to collagen protein.Being called collagen protein at this hydrolytic collagen, is to want to distinguish to some extent with collagen, collagen and collagen protein is not lumped together.In " collagen protein " (Chemical Industry Press, calendar year 2001) book, collagen, gelatin and collagen protein have been done preliminary defining, not too distinct or not too attractive definite conception originally.The research work of Li Guoying etc. recently, (Li Guoying etc. the performance difference of collagen, gelatin and Collagen Hydrolysate.Sichuan University's journal (engineering science version), 2005,37 (4): 54～58) further confirmed difference between them on three's the performance.
Collagen is meant the class protein that exists in animal tissues's organ, is extracting, when separating, along with method is different with condition, can produce collagen, gelatin and three kinds of products of collagen protein.Can be called as collagen, must be that proteinoid that triple-helix structure does not change, and also remains with biological activity.Gelatin is the denatured products of collagen under acid, alkali, enzyme or high temperature action, and is the same with collagen, is made up of 18 seed amino acids, but lost biological activity.Collagen becomes gelatin three kinds of possibilities: 1. the triple helix body is totally released, and becomes three mutual unconnected crumpled peptide chains, and their composition and relative molecular mass have nothing in common with each other; 2. a peptide chain is totally released, and the covalent linkage between the two other peptide chain all disconnects, but still has hydrogen bond to connect; 3. still have a small amount of hydrogen bond to be bound up after three peptide chains unclamp.
Say on the wide significance that gelatin also belongs to collagen protein, just the molecular weight ratio collagen protein of gelatin is higher, and also has a small amount of hydrogen bond between the peptide chain of gelatin.On structure, collagen is to have the protein that covalent linkage connects three strands of α-peptide chains, gelatin is that covalent linkage is interrupted substantially, the protein of surplus secondary key only, and collagen protein has been basically not by covalent linkage and the constraint of secondary key, the α-peptide chain or the segment of gelatin molecule more freely.
On performance, collagen can form film, and film has snappiness, elasticity and intensity preferably.Under the simulation physiological condition, collagen solution is placed 25min, can observe between the tropocollagen molecule and can be interconnected to form fiber once again from the variation of its relevant absorbancy, causes absorbancy to rise; The denatured products gelatin of collagen and degraded product collagen protein then do not have such character.The triple-helix structure of gelatin and collagen protein is all destroyed, and this destruction is irreversible, thereby has lost fibering again.Gelatin solution can form gel, and heating is liquefaction then, drops to below 30 ℃ can become gel again; Gelatin also can film forming, but gelatin film is more crisp, and the strength ratio collagem membrane is poor; Collagen protein can not film forming.The cell growth experiment proves, collagen protein is the same with gelatin not to have the character that promotes the cell growth, do not have a biological activity.
The research that collagen class material is extracted abroad starts from eighties of last century forties, and the research that China extracts collagen class material more early.But practical at present extracting method relates to the method with enzymic hydrolysis more.Therefore the material collagen protein that extracts mixes mutually with gelatin, rather than collagen.And how not possess biological activity, or with the biological activity of non-collagen class material, being used as is the biological activity of collagen.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of antler collagen efficiently, its be with the pilose antler homogenate earlier after neutrality, acidic buffer, ethanolic soln repeatedly after the lixiviate, again through refluxing extraction, and dialyse, freeze-drying, thereby obtain bright relatively pilose antler, yield is about 10～15% antler collagen.Prove that through cell experiment this collagen has proliferation function to rat osteoblast.
Preparation method of the present invention comprises the steps:
1. pilose antler pre-treatment: get fresh pilose antler, peeling, fleshing is divided into the long segment of 8～10cm, and 1～4 ℃ is hung static 24～36h, and the blood control is clean, is cut into 0.5～1cm
3Fritter, 1～4 ℃ of distilled water is washed till no color;
2. remove water-soluble protein: high-speed tissue mashing machine's homogenate pilose antler adds Tris-HCl damping fluid (or the NaH of pH7.5～8.3 of 15～25mM pH7.5～8.3 that contain 1～3mM beta-mercaptoethanol and 1～3mM EDTA of 1.5～2 times of homogenate volumes
2PO
4-Na
2HPO
4Damping fluid), stir, 1～4 ℃ of lixiviate 24～36h, centrifugal 30～the 50min of 4000～5000rpm gets supernatant liquor and precipitation, precipitation adds the above-mentioned damping fluid of 1～1.5 times of its volume again, stirs 1～4 ℃ of lixiviate 24～36h, centrifugal 30～the 50min of 4000～5000rpm must remove the impurity-removing precipitating of water-soluble protein;
Merge the lixiviate supernatant liquor and can be used for preparing water-soluble pilose antler albumen;
3. the extraction of collagen: get the impurity-removing precipitating that above-mentioned steps obtains, clean with 10～15mM, pH7.1～7.5Tris-HCl damping fluid, 20～30mM the pH3.1 that contains 1～3mM beta-mercaptoethanol and the 1～3mMEDTA～3.5 HOAc-NaOAc damping fluids that add 1～1.5 times of precipitation volume again, stir, 1～4 ℃ of lixiviate 48～72h, centrifugal 30～the 50min of 4000～5000rpm abandons supernatant; Precipitation is cleaned with 10～15mM pH7.1～7.5 Tris-HCl damping fluids, adds 0～4 ℃ of lixiviate 36～48h of 65%～70% ethanol of 1.5～2 times of volumes of precipitation volume, 60～65 ℃ of refluxing extraction 48～72h;
4. reclaim ethanol: 50～55 ℃, 60～65rpm rotary evaporation recovery ethanol, extracting solution places the dialysis tubing of the molecular weight ranges 8～15kDa that dams, to distill water dialysis 24～36h ,-20 ℃ of pre-freeze 24～48h ,-50～-55 ℃ of freeze-drying promptly get antler collagen.
The advantage that the present invention has is the more existing collagen extracting method of the extraction process of antler collagen, and is easy, easily row; And, bright relatively pilose antler, the yield height, the purity height, and can be used for amplifying the characteristics of production.Pilot process can also obtain can be used in the general extractive that extracts the pilose antler water-soluble protein, and efficient is higher.The gained sample is in the SDS-PAGE polyacrylamide gel electrophoresis, and the molecular weight distribution of antler collagen concentrates on two portions, and a part is two protein bands that molecular weight is close between 100kDa-200kDa; Another part is more than 200kDa; Activity experiment shows that antler collagen has the effect that promotes the cell growth, still maintains biological activity.Prove that our extracting method does not destroy the native conformation of collagen.
Description of drawings
SDS～PAGE the electrophorogram of the antler collagen of Fig. 1: embodiment 1 preparation;
SDS～PAGE the electrophorogram of the pilose antler water-soluble protein of Fig. 2: embodiment 1 preparation.
As shown in Figure 1, right side, antler collagen; The left side, the high molecular standard protein is 200kDa from top to bottom successively, 105kDa, 79kDa, 51kDa, 31kDa.
As shown in Figure 2, M is a molecular weight standard albumen, and molecular weight is followed successively by 97KD from top to bottom, 66KD, 43KD, 31KD, 20KD, 14KD; DAP is the water-soluble total protein of pilose antler.
Embodiment
Embodiment 1:
1. pilose antler pre-treatment: get fresh pilose antler 3.0kg, peeling, fleshing is divided into the long segment of 8cm, and 4 ℃ are hung static 24h, and the blood control is clean, are cut into 1cm
3Fritter, 4 ℃ of distilled water are washed till no color.
2. remove water-soluble protein: high-speed tissue mashing machine's homogenate, get pilose antler homogenate 1.5L, add the 20mM pH8.0 Tris-HCl damping fluid that 3.0L contains 1mM beta-mercaptoethanol and 1mM EDTA again, stir, 4 ℃ of lixiviate 24h, the centrifugal 30min of 4500rpm must precipitate 2.5L, adds the above-mentioned damping fluid of 3L again, stir, 4 ℃ of lixiviate 24h, the centrifugal 30min of 4500rpm must remove the impurity-removing precipitating 2.5L of water-soluble protein.
Merge the lixiviate supernatant liquor twice, behind 0.45 μ m water system filter membrane suction filtration, with supernatant 100% ammonium sulfate precipitation, 4 ℃ of standing over night, the gained precipitation adds 9L, 20mM pH7.4 Tris-HCl damping fluid renaturation, and (6000～8000Da) spend the night, and dialysis keeps liquid-20 ℃ pre-freeze 24h in 4 ℃ of dialysis,-55 ℃ of freeze-drying obtain the about 3g of water-soluble pilose antler total protein;
3. the extraction of collagen: remove the impurity-removing precipitating 2.5L that removes water-soluble total protein, after cleaning with the Tris-HCl damping fluid of 5L, 10mM, pH7.4, add the 20mMpH3.3 HOAc-NaOAc damping fluid that 3.5L contains 1mM beta-mercaptoethanol and 1mM EDTA again, stir, 4 ℃ of lixiviate 48h, the centrifugal 30min of 4500rpm abandons supernatant, must precipitate 2.5L; Clean with 10mM pH7.4 Tris-HCl damping fluid, add 4 ℃ of lixiviate 48h of 4.5L 70% ethanol, 60 ℃ of refluxing extraction 56h;
4. reclaim ethanol: 50 ℃, the 60rpm rotary evaporation reclaims ethanol, extracting solution places the dialysis tubing of the molecular weight ranges 8～15kDa that dams, to distill water dialysis 24h, dialysis keeps liquid in-20 ℃ of pre-freeze 24h, in-55 ℃ of freeze-drying, promptly get antler collagen 0.3kg again, range of molecular weight distributions is more than 100,000.
Table 1: the amino acid composition analysis of this routine gained antler collagen
| Amino acid | Content (%) | Amino acid | Content (%) |
| Glycine Gly proline(Pro) Pro L-Ala Ala oxyproline q-Pro L-glutamic acid Glu aspartic acid Asp arginine Arg Serine Ser Methionin Lys | 32.24 12.64 10.78 10.20 8.46 4.55 4.42 3.56 2.94 | Leucine Leu Threonine Thr Xie Ansuan Val phenylalanine Phe Isoleucine Ile Histidine His tyrosine Tyr methionine(Met) Met Gelucystine Cys | 2.59 2.33 2.22 1.22 1.08 0.57 0.21 - - |
Annotate:, adopt Agilent company 1100 series of high efficiency liquid phases to carry out aminoacids content and measure by test center of Jilin University.
By table 1 as seen in 20 kinds of common amino acids, glycine, proline(Pro) and L-Ala content (mass ratio) are the highest, wherein glycine content has almost accounted for 1/3, is 32.24%, and proline(Pro), L-Ala and hydroxyproline content are respectively 12.64%, 10.78% and 10.20%; Histidine and tyrosine content are lower, do not detect Gelucystine; Conform to the collagen amino acid characteristics of document (Jiang Tingda chief editor, collagen and collagen protein [M], Beijing: Chemical Industry Press, 2006.1) report, illustrate that the pilose antler alcohol extract is mainly collagen class material.
In the extraction of collagen, sepn process, because method is different with condition, can produce collagen, gelatin and three kinds of products of collagen protein, there is bigger difference in they on structure and performance.Can be called as collagen, must be that proteinoid that its triple-helix structure does not change, and relative molecular mass is approximately 300,000, and to distribute very narrow; Gelatin is the hydrolysate of collagen under acid, alkali, enzyme or high temperature action, relative molecular mass from several thousand to 100,000, and it is very wide to distribute; Collagen protein is the violent hydrolysate of collagen or gelatin, is polypeptide mixture, relative molecular mass from several thousand to several ten thousand, and molecular weight distribution is also very wide.By the SDS-PAGE electrophoresis method, we adopt the proteinic range of molecular weight distributions of pure extracting method preparation in 100,000 above (see figure 1)s.
So far found 19 kinds of different collagen, these collagens have certain specificity in in-house distribution, are mainly type i collagen in skin and the osseous tissue.Each Collagen Type VI all contains oxyproline and hydroxylysine, and the oxyproline of each Collagen Type VI is different with the ratio of proline(Pro), and type i collagen is 0.7～0.8.This ratio of the antler collagen that we extract is 0.8, this shows that the alcohol extracting antler collagen should be type i collagen.This result and bibliographical information (Jiang Tingda chief editor, collagen and collagen protein [M], Beijing: Chemical Industry Press, 2006.1) unanimity.Type i collagen mainly is made up of 3 α 1 (I) chain, and promptly α 1 (I) 3 also has the part type i collagen to be made up of two α 1 (I) chains and a α 2 (I) chain, i.e. α 1 (I) 2 α 2 (I).Every peptide chain has 1000 left and right sides amino-acid residues, and about relative molecular mass 100kDa, the molecular weight of α 1 is slightly larger than α 2.Because of lacking halfcystine in the tropocollagen molecule amino acid composition, therefore, can not as other albumen, link between every chain with disulfide linkage, but by the covalent cross-linking between Histidine and Methionin.Therefore, collagen can not depolymerized to strand because of the existence of beta-mercaptoethanol (open the disulfide linkage in the protein, make protein deaggregation) in the SDS-PAGE electrophoresis, detect molecular weight and should be about 300kDa.But can see in this experiment, between 100kDa-200kDa, have two protein bands that molecular weight is close, should be α 1 (I) chain and α 2 (I) chain of type i collagen.This may be because when carrying out the SDS-PAGE electrophoresis, and for protein chain being stretched as far as possible and fully combining with SDS, to the way that sample has adopted boiling water to heat, this step may cause the partial hydrolysis of collagen; Also may be in the collagen leaching process, partial hydrolysis has taken place.In case collagen generation hydrolysis just the corresponding proteins band can occur in the SDS-PAGE electrophoresis.It should be noted that no matter this hydrolysis all should be comparatively gentle owing to which kind of reason has caused the collagen hydrolysis.This is because on electrophoresis result, the protein band of molecular weight less than 100kDa do not occur, show that hydrolysis only causes the release of α 1 (I) chain and α 2 (I) chain, and every strand itself is hydrolyzed not, and structure is all very complete.In addition, the molecular weight of antler collagen α 1 (I) chain and α 2 (I) chain is slightly larger than bibliographical information, may cause owing to tissue-derived difference.
Claims (5)
1. the preparation method for separating and purifying of antler collagen, its step is as follows:
1. remove water-soluble protein: high-speed tissue mashing machine's homogenate pilose antler adds the Tris-HCl damping fluid of 15～25mM pH7.5～8.3 that contain 1～3mM beta-mercaptoethanol and 1～3mM EDTA of 1.5～2 times of homogenate volumes or the NaH of pH7.5～8.3
2PO
4-Na
2HPO
4Damping fluid, stirring, lixiviate, centrifugal supernatant liquor and the precipitation of getting, precipitation adds the above-mentioned damping fluid of 1～1.5 times of its volume again, stirs lixiviate, the centrifugal impurity-removing precipitating that must remove water-soluble protein;
2. the extraction of collagen: the impurity-removing precipitating of getting the above-mentioned steps acquisition is cleaned, 20～30mM pH3.1～3.5HOAc-NaOAc the damping fluid that contains 1～3mM beta-mercaptoethanol and 1～3mM EDTA that adds 1～1.5 times of precipitation volume again, stir, 1～4 ℃ of lixiviate 48～72h, centrifugal 30～the 50min of 4000～5000rpm abandons supernatant; 0～4 ℃ of lixiviate 36～48h of 65%～70% ethanol that adds 1.5～2 times of volumes of precipitation volume after precipitation is cleaned, 60～65 ℃ of refluxing extraction 48～72h;
3. reclaim ethanol: 50～55 ℃, 60～65rpm rotary evaporation recovery ethanol, extracting solution promptly gets antler collagen after dialysis, pre-freeze, freeze-drying.
2. the preparation method for separating and purifying of antler collagen as claimed in claim 1 is characterized in that: pilose antler is handled the back and is used, and it is with fresh pilose antler peeling, fleshing is divided into the long segment of 8～10cm, and 1～4 ℃ is hung static 24～36h, the blood control is clean, be cut into 0.5～1cm
3Fritter, 1～4 ℃ of distilled water is washed till no color.
3. the preparation method for separating and purifying of antler collagen as claimed in claim 1, it is characterized in that: 1. step is in 1～4 ℃ of lixiviate 24～36h, in the centrifugal 30～50min of 4000～5000rpm.
4. the preparation method for separating and purifying of antler collagen as claimed in claim 1 is characterized in that: step 2. impurity-removing precipitating is to clean with 10～15mM, pH7.1～7.5 Tris-HCl damping fluids.
5. the preparation method for separating and purifying of antler collagen as claimed in claim 1, it is characterized in that: step 3. gained extracting solution is the dialysis tubing that places the molecular weight ranges 8～15kDa that dams, to distill water dialysis 24～36h ,-20 ℃ of pre-freeze 24～48h ,-50～-55 ℃ of freeze-drying get antler collagen.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103408634A (en) * | 2013-07-16 | 2013-11-27 | 新疆医科大学 | Horse placenta water-soluble protein extract, preparation method and application thereof |
| CN103769393A (en) * | 2012-10-19 | 2014-05-07 | 中国科学院大连化学物理研究所 | Cleaning method for completely removing blood residues in pilose antler |
| CN106333966A (en) * | 2015-07-13 | 2017-01-18 | 中国中医科学院医学实验中心 | Preparation method of corn cervi pantotrichum extract, and related products |
| CN109485692A (en) * | 2018-12-06 | 2019-03-19 | 吉林农业大学 | A kind of method of activated protein in extraction pilose antler |
-
2007
- 2007-12-12 CN CNA2007101935410A patent/CN101182358A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103769393A (en) * | 2012-10-19 | 2014-05-07 | 中国科学院大连化学物理研究所 | Cleaning method for completely removing blood residues in pilose antler |
| CN103769393B (en) * | 2012-10-19 | 2016-06-01 | 中国科学院大连化学物理研究所 | A kind of purging method that can thoroughly remove blood residuals in pilose antler |
| CN103408634A (en) * | 2013-07-16 | 2013-11-27 | 新疆医科大学 | Horse placenta water-soluble protein extract, preparation method and application thereof |
| CN103408634B (en) * | 2013-07-16 | 2016-04-13 | 新疆医科大学 | Horse placenta water-soluble protein extract and its preparation method and application |
| CN106333966A (en) * | 2015-07-13 | 2017-01-18 | 中国中医科学院医学实验中心 | Preparation method of corn cervi pantotrichum extract, and related products |
| CN109485692A (en) * | 2018-12-06 | 2019-03-19 | 吉林农业大学 | A kind of method of activated protein in extraction pilose antler |
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