CN103146789B - Preparation method of collagen - Google Patents
Preparation method of collagen Download PDFInfo
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- CN103146789B CN103146789B CN201310065180.7A CN201310065180A CN103146789B CN 103146789 B CN103146789 B CN 103146789B CN 201310065180 A CN201310065180 A CN 201310065180A CN 103146789 B CN103146789 B CN 103146789B
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Abstract
The invention provides a preparation method of collagen. The preparation method is characterized in that earthworm serine protease creatively utilized as an enzymic method is used for preparing zymogen of the collagen. The hydrolysis efficiency is remarkably improved; an ultrafiltration technology is added in the preparation process, so that an enzymatic hydrolysate is thoroughly separated, the collagen has uniformly distributed small molecular weights and high activity and can be easily absorbed by a human body. The whole process course of the preparation method of the collagen is mild in condition and environmentally-friendly.
Description
Technical field
The present invention relates to the preparation invention of biologically active substance, refer to especially a kind of preparation method of collagen protein.
Background technology
Collagen protein is the protein that people's in-vivo content is maximum, account for 1/3rd of body internal protein total amount, account for human body weight 6%. it be the chief component of reticular tissue, mainly exist with the form of collegen filament, mainly be present in skin, muscle, bone, tooth, internal organ are (as stomach, intestines, heart and brain, blood vessel and esophagus) with the position such as eyes. it is (crease-resistant that collagen protein has beauty treatment, moisturizing, body shaping), ensure motor system (bone, muscle and joint) and cardio-cerebrovascular health, regulation and control internal secretion, maintenance internal organs, improve the multi-efficiencies such as body immunity. in addition, because collagen protein also has good biocompatibility, trophicity, reparation property, moisture retention, compatibleness and affinity, so can be widely used in biomedical material, makeup, the growth of collagen protein and human body in the functional product such as food and healthcare products, senescence and disease has extremely close contact, to protect and help people's important nutrient in all one's life.
The mode of production of protein hydrolyzate has two kinds conventionally: chemical degradation method and enzyme liberating method.Chemical method is to impel at a certain temperature the Fragmentation of protein molecule to form the method for small-molecule substance with chemical reagent such as acid, alkali.Acid system adopts the strong acid such as hydrochloric acid, sulfuric acid at high temperature to react more, strong reaction, and equipment corrosion is serious, and hydrolysis is thorough, the peptide molecule non-uniform mass obtaining, tryptophane is completely destroyed, and this method is eliminated at present gradually; Alkali process hydrolysis can be because the transition stage of unsymmetrical carbon process intermediateness, there is racemization phenomenon, produce D type and L-type etc. molar mixture, make the most racemization of amino acid, wherein, D-type amino acid can not be served as effective amino acid source, and some also has toxicity and even has carcinogenic, teratogenesis and mutagenesis.The eighties in 20th century, along with the fast development of Enzymes Industry and foodstuffs industry, it is found that enzymatic hydrolysis reaction conditions gentleness, reaction times is short, non-environmental-pollution, product is of high nutritive value, and product is taking polypeptide and L-type total free aminoacids as main, be easy to human body and digest and assimilate, thereby utilize enzymic hydrolysis collagen protein to be subject to the people's attention gradually.
Can make the enzyme of collagen protein enzymolysis more.Can be divided into restriction endonuclease and excision enzyme according to action site; From originating, can be divided into plant protease (as bromeline, papoid etc.), animal protease (as trypsinase, stomach en-etc.), microbial protease (as Bacillus subtilus 1398, actinomycetes 166 etc.); In addition, be usually used in hydrolysis proteolytic enzyme also have local flavor prozyme etc.In actual applications, choosing of enzyme will be considered three aspects conventionally: the one, and the intensity of enzyme to collagen protein effect; The 2nd, the price of enzyme; The 3rd, the requirement of hydrolysate.If enzyme to the effect too of collagen a little less than, cannot obtain high collagen percent hydrolysis, in addition, also must consider the action site of enzyme to collagen, because this directly affects the distribution of last hydrolysate molecular weight, it is a key factor that determines to obtain target product, the at present known enzyme class about preparing collagen protein is also very many, but its hydrolysis efficiency is not generally very high, therefore, on hydrolysis efficiency, also need to find that a kind of new enzyme is further to improve.
Earthworm serine protease is to extract a kind of aspartic protease obtaining in earthworm body, belongs to trypsinase, its energy protein hydrolysate substrate (as casein), the ester of basic aminoacids and acid amides.PI3-4; 20-50 DEG C of scope is stable; It is a kind of glycoprotein enzyme.
Earthworm serine protease is as a histone lytic enzyme, deep not enough to the research of its zymologic property.Most earthworm serine protease components are subject to the inhibition of 3 kinds of serpin phenylmethylsulfonyl fluorides, tolylsulfonyl phenylalanyl chloromethyl ketone, N-tolysulfonyl lysyl chloromethyl ketone, but different components there are differences the susceptibility of different types of proteinase inhibitor; Earthworm serine protease directly cut Profibrinolysin and fibrin degradation former be its molecular basis simultaneously with kinases and plasmin activity; The synthetic substrate of difference is had to specificity and illustrate that it has different activities center; Earthworm serine protease can have hydrolytic action to the specificity substrate S-2288 of tissue plasminogen activator (t-P A), illustrates that it is similar to t-P A to the active mode of Profibrinolysin.
Summary of the invention
The present invention proposes a kind of preparation method of collagen protein, has improved the efficiency of hydrolysis, and efficiency of pcr product is higher, and the content of the peptide simultaneously obtaining is higher, and product separation is more thorough, active high.
Technical scheme of the present invention is achieved in that a kind of preparation method of collagen protein, comprises the following steps:
(1) pre-treatment: the raw material of preparing collagen protein is cleaned up, then use 3 ‰-10 ‰ basic solution to soak 10-20min, then raw material is cleaned to neutral, dry stand-by;
(2) take a certain amount of pretreated raw material, add purified water according to material ratio 1:3 (w/v)-1:4 (w/v), 80-85 DEG C of sterilizing 30-40min, is cooled to 40 DEG C;
(3) add earthworm serine protease by the 0.5-2 ‰ that takes pretreated raw material weight;
(4) enzymolysis process control: by buffered soln, PH is controlled in the scope of 6.0-7.8,37-42 DEG C of insulation hydrolysis 6-7h, 80-85 DEG C of deactivation 30min, then centrifuging, got cleaner liquid;
(5) to use molecular weight cut-off be 6000, and ultrafiltration apparatus carries out ultrafiltration, and adds purified water and make ultrafiltration more complete, gets peritoneal effluent;
(6) peritoneal effluent is dried, obtains collagen protein.
Preferably, in step (1), the NaOH solution that described basic solution is 5 ‰.
Preferably, in step (2), sterilising temp is 80 DEG C of 30min, and excess Temperature easily causes protein denaturation, reduces the value of finished product.
Preferably, in step (3), the amount that adds earthworm serine protease is to take 1 ‰ of pretreated raw material weight.
Preferably, in step (4), PH is controlled at 6.5, and the temperature of insulation hydrolysis is 40 DEG C, hydrolysis time 6.5 hours, and deactivation temperature is 80 DEG C.
Preferably, in step (5), described ultrafiltration apparatus is hollow-fibre membrane or rolled film or tubular membrane or ceramic membrane, and certainly, available ultrafiltration apparatus is not limited to this, as long as can reach the ultrafiltration apparatus of effect same, all can use.
Preferably, in step (6), described be dried into lyophilize or spraying dry, adopt that lyophilize or spraying are dry can prevent collagen protein generation sex change.
Preferably, in step (1), described raw material is skin or the hoof of fish-skin, fish scale or bird, domestic animals, and the selection of described raw material is not limited to this, is the different selection difference due to raw material itself, and the yield of product can be slightly different.
Beneficial effect of the present invention is: the earthworm serine protease that utilizes of the invention is prepared the proenzyme of collagen protein as enzyme process, its hydrolysis efficiency significantly improves, these can other data such as peptide content (reaching more than 80%) from hydrolysis find out, it is apparently higher than other known enzymes; In the present invention, also by adding and use ultra-filtration technique in preparation link, enzymolysis product is separated thoroughly, collagen molecules amount is uniformly distributed, and the following component of 3000 molecular weight reaches more than 90%, and molecular weight is little, active high, is easier to absorption of human body; The preparation method's of a kind of collagen protein of the present invention whole technical conditions gentleness, environmentally friendly.
Embodiment
For understanding better the present invention; below by following examples, the present invention is done to further concrete elaboration; but unintelligible is limitation of the invention; the foregoing invention content of some nonessential improvement and adjustment do according to to(for) those skilled in the art, be also considered as dropping in protection scope of the present invention.
We adopt method disclosed by the invention, carry out following operation:
(1) pre-treatment: after fish-skin water is cleaned up, use 5 ‰ NaOH solution soaking 15min, fish-skin is cleaned to till neutrality, dry stand-by;
(2) take pretreated fish-skin 500g, by material ratio 1:3(w/v) add 1500ml purified water, 80 DEG C of sterilizing 30min, are cooled to 40 DEG C;
(3) add earthworm serine protease 0.5g(source by the pretreated fish-skin weight 1 ‰ taking: Zhuhai Bokang Pharmaceutical Industry Co., Ltd.);
(4) PH is controlled at 6.5,40 DEG C of insulation hydrolysis 6.5h, 80 DEG C of deactivation 30min, and centrifuging, obtains 1200ml solution;
(5) use the tubular fibre membrane ultrafiltration that molecular weight cut-off is 6000, centre is added 1200ml purified water again, finally obtains peritoneal effluent 2100ml,
(6) ultrafiltration peritoneal effluent carries out lyophilize.
Result: the product obtaining is detected to analysis, obtain collagen protein 36.55g, weight recovery is 7.31%.Wherein, peptide content is that the following component of 80.66%, 3000 molecular weight reaches more than 90% especially; Oxyproline content is 9.5%; As shown in the table with the distribution of gel chromatography detection molecules amount and content results:
| Crest segment | Number-average molecular weight Mn | Weight-average molecular weight Mw | Wide D distributes | Relative content (%) |
| 1# | 1.62×10 4 | 1.66×10 4 | 1.03 | 13.2 |
| 2# | 9.16×10 3 | 9.17×10 3 | 1.00 | 6.37 |
| 3# | 6.06×10 3 | 6.11×10 3 | 1.01 | 31.2 |
| 4# | 3.22×10 3 | 3.26×10 3 | 1.01 | 45.6 |
| 5# | 1.01×10 3 | 1.027×10 3 | 1.01 | 3.59 |
| Always | 4.04×10 3 | 6.26×10 3 | 1.55 | 100 |
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, within the spirit and principles in the present invention not all, any amendment of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (8)
1. a preparation method for collagen protein, is characterized in that, comprises the following steps:
(1) pre-treatment: the raw material of preparing collagen protein is cleaned up, then use 3 ‰-10 ‰ basic solution to soak 10-20min, then raw material is cleaned to neutral, dry stand-by;
(2) take a certain amount of pretreated raw material, add purified water according to material ratio 1:3 (w/v)-1:4 (w/v), 80-85 DEG C of sterilizing 30-40min, is cooled to 40 DEG C;
(3) add earthworm serine protease by the 0.5-2 ‰ that takes pretreated raw material weight;
(4) enzymolysis process condition control: PH is controlled at 6.0-7.8,37-42 DEG C of insulation hydrolysis 6-7h, 80-85 DEG C of deactivation 30min, then centrifuging, got cleaner liquid;
(5) to use molecular weight cut-off be 6000, and ultrafiltration apparatus carries out ultrafiltration, and adds purified water and make ultrafiltration more complete, gets peritoneal effluent;
(6) peritoneal effluent is dried, obtains collagen protein.
2. the preparation method of collagen protein as described in claim 1, is characterized in that: in step (1), and the NaOH solution that described basic solution is 5 ‰.
3. the preparation method of collagen protein as described in claim 1, is characterized in that: in step (2), sterilising temp is 80 DEG C, 30min.
4. the preparation method of collagen protein as described in claim 1, is characterized in that: in step (3), the amount that adds earthworm serine protease is to take 1 ‰ of pretreated raw material weight.
5. the preparation method of collagen protein as described in claim 1, is characterized in that: in step (4), PH is controlled at 6.5, and the temperature of insulation hydrolysis is 40 DEG C, hydrolysis time 6.5 hours, and deactivation temperature is 80 DEG C.
6. the preparation method of collagen protein as described in claim 1, is characterized in that: in step (5), described ultrafiltration apparatus is hollow-fibre membrane or rolled film or tubular membrane or ceramic membrane.
7. the preparation method of collagen protein as described in claim 1, is characterized in that: in step (6), described dry employing lyophilize or spraying are dry.
8. the preparation method of collagen protein as described in claim 1, is characterized in that: in step (1), described raw material is skin or the hoof of fish-skin, fish scale or bird, domestic animals.
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| CN201310065180.7A CN103146789B (en) | 2013-02-28 | 2013-02-28 | Preparation method of collagen |
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| CN103146789B true CN103146789B (en) | 2014-11-26 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104017072A (en) * | 2014-06-18 | 2014-09-03 | 常州药物研究所有限公司 | Complete device for collagen purification |
| WO2016076276A1 (en) * | 2014-11-11 | 2016-05-19 | 株式会社ニッピ | Immunopotentiator, cell-mediated immunopotentiator, and t cell proliferation agent |
| WO2016172894A1 (en) * | 2015-04-30 | 2016-11-03 | 上海欣吉特生物科技有限公司 | Inactivated collagen material and preparation method thereof |
| CN107177658A (en) * | 2017-07-28 | 2017-09-19 | 美泰科技（青岛）股份有限公司 | A kind of preparation method of Cartilage collagen peptide |
| CN107794290A (en) * | 2017-11-29 | 2018-03-13 | 桂林华诺威生物科技有限公司 | The preparation method of one seed oyster collagen |
| CN110810852A (en) * | 2019-11-04 | 2020-02-21 | 江西沐恩堂生物科技有限公司 | Preparation method of earthworm freeze-dried powder for regulating cardiovascular function |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921821A (en) * | 2010-08-06 | 2010-12-22 | 郁小兵 | Method for extracting high-quality collagen at low temperature |
| CN102154424A (en) * | 2011-01-14 | 2011-08-17 | 国家海洋局第三海洋研究所 | Process for preparing micro-molecular fish scale collagen peptides |
| CN102690853A (en) * | 2012-03-22 | 2012-09-26 | 浙江省海洋开发研究院 | Preparation method of sleeve-fish skin low-molecular-weight collagen peptide |
| CN102703555A (en) * | 2012-06-08 | 2012-10-03 | 集美大学 | Extraction and preparation method for micromolecule fishskin collagen peptide |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101921821A (en) * | 2010-08-06 | 2010-12-22 | 郁小兵 | Method for extracting high-quality collagen at low temperature |
| CN102154424A (en) * | 2011-01-14 | 2011-08-17 | 国家海洋局第三海洋研究所 | Process for preparing micro-molecular fish scale collagen peptides |
| CN102690853A (en) * | 2012-03-22 | 2012-09-26 | 浙江省海洋开发研究院 | Preparation method of sleeve-fish skin low-molecular-weight collagen peptide |
| CN102703555A (en) * | 2012-06-08 | 2012-10-03 | 集美大学 | Extraction and preparation method for micromolecule fishskin collagen peptide |
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