CN103146789A - Preparation method of collagen - Google Patents
Preparation method of collagen Download PDFInfo
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- CN103146789A CN103146789A CN2013100651807A CN201310065180A CN103146789A CN 103146789 A CN103146789 A CN 103146789A CN 2013100651807 A CN2013100651807 A CN 2013100651807A CN 201310065180 A CN201310065180 A CN 201310065180A CN 103146789 A CN103146789 A CN 103146789A
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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 maximum protein of people's in-vivo content, 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 the form with collegen filament exists, 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, the regulation and control internal secretion, the 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 usually: 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 that obtains, tryptophane is fully destroyed, and this method is eliminated at present gradually; Alkali process hydrolysis can be because the transition stage of unsymmetrical carbon process intermediateness, the racemization phenomenon occurs, namely produce D type and L-type etc. molar mixture, make the most racemization of amino acid, wherein, D-type amino acid can not be as effective amino acid source, and some also has toxicity carcinogenic, teratogenesis and mutagenesis are even arranged.The eighties in 20th century, fast development along with Enzymes Industry and foodstuffs industry, it is found that the enzymatic hydrolysis reaction conditions is gentle, reaction times is short, non-environmental-pollution, product is of high nutritive value, and product is take polypeptide and L-type total free aminoacids as main, be easy to human body and digest and assimilate, thereby utilize the 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; 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.) on originating; In addition, the proteolytic enzyme that is usually used in being hydrolyzed also has local flavor prozyme etc.In actual applications, choosing of enzyme will be considered three aspects usually: the one, and the intensity of enzyme to the collagen protein effect; The 2nd, the price of enzyme; The 3rd, the requirement of hydrolysate.If a little less than the effect too of enzyme to collagen, can't obtain high collagen percent hydrolysis, in addition, must consider that also enzyme is to the action site of 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 the preparation collagen protein is also very many, but its hydrolysis efficiency is not generally very high, therefore, on hydrolysis efficiency, need also to find that a kind of new enzyme is with further raising.
The earthworm serine protease is to extract a kind of aspartic protease that obtains in the earthworm body, belongs to trypsinase, its energy protein hydrolysate substrate (as casein), the ester of basic aminoacids and acid amides.PI3-4; 20-50 ℃ of scope is stable; It is a kind of glycoprotein enzyme.
The earthworm serine protease is as a histone lytic enzyme, and is deep not enough to the research of its zymologic property.Most earthworm serine protease components are subjected 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; The earthworm serine protease directly cut Profibrinolysin and fibrin degradation former be its molecular basis that has simultaneously kinases and plasmin activity; The synthetic substrate of difference is had specificity illustrate that it has the different activities center; The earthworm serine protease can have hydrolytic action to the specificity substrate S-2288 of tissue plasminogen activator (t-P A), illustrates that its active mode to Profibrinolysin is similar to t-P A.
Summary of the invention
The present invention proposes a kind of preparation method of collagen protein, has improved the efficient of hydrolysis, and efficiency of pcr product is higher, and the content of the peptide that obtains simultaneously is higher, and product separation is more thorough, and is 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 that will prepare collagen protein cleans up, and then uses 3 ‰-10 ‰ basic solution to soak 10-20min, then raw material is cleaned to neutral, dries 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 ℃ of sterilization 30-40min is cooled to 40 ℃;
(3) add the earthworm serine protease by the 0.5-2 ‰ that takes pretreated raw material weight;
(4) enzymolysis process is controlled: make PH be controlled in the scope of 6.0-7.8 by buffered soln, and 37-42 ℃ of insulation hydrolysis 6-7h, 80-85 ℃ of deactivation 30min, cleaner liquid was got in then centrifuging;
(5) using molecular weight cut-off is that 6000 ultrafiltration apparatus carries out ultrafiltration, and adds purified water and make ultrafiltration more complete, gets peritoneal effluent;
(6) peritoneal effluent is carried out drying, obtain collagen protein.
Preferably, in step (1), described basic solution is 5 ‰ NaOH solution.
Preferably, in step (2), sterilising temp is 80 ℃ of 30min, and excess Temperature easily causes protein denaturation, reduces the value of finished product.
Preferably, in step (3), the amount that adds the 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 ℃, hydrolysis time 6.5 hours, and the deactivation temperature is 80 ℃.
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 drying is lyophilize or spraying drying, adopts lyophilize or spraying drying 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, be different selection due to the difference of raw material itself, the yield of product can be slightly different.
Beneficial effect of the present invention is: the earthworm serine protease that utilizes of the invention prepares the proenzyme of collagen protein as enzyme process, its hydrolysis efficiency significantly improves, these can from the hydrolysis after other data such as peptide content (reaching more than 80%) find out, it is apparently higher than other known enzymes; Also use ultra-filtration technique by adding in the preparation link in the present invention, enzymolysis product is separated thoroughly, the collagen molecules amount evenly distributes, and the 3000 following components of molecular weight reach more than 90%, and molecular weight is little, and is 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 is gentle, environmentally friendly.
Embodiment
For understanding better the present invention; below by following examples, the present invention is done further concrete elaboration; but unintelligible is limitation of the invention; some nonessential improvement and adjustment for those skilled in the art does according to the foregoing invention content also are 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 the fish-skin water is cleaned up, use 5 ‰ NaOH solution soaking 15min, fish-skin is cleaned till the neutrality, dry stand-by;
(2) take pretreated fish-skin 500g, by material ratio 1:3(w/v) add the 1500ml purified water, 80 ℃ of sterilization 30min are cooled to 40 ℃;
(3) add earthworm serine protease 0.5g(source by the pretreated fish-skin weight 1 ‰ that takes: Zhuhai Bokang Pharmaceutical Industry Co., Ltd.);
(4) PH is controlled at 6.5,40 ℃ of insulation hydrolysis 6.5h, 80 ℃ of deactivation 30min, and centrifuging gets 1200ml solution;
(5) the use molecular weight cut-off is 6000 tubular fibre membrane ultrafiltration, and the centre is added the 1200ml purified water again, gets at last peritoneal effluent 2100ml,
(6) the ultrafiltration peritoneal effluent carries out lyophilize.
Result: the product that obtains is detected analysis, get collagen protein 36.55g, weight recovery is 7.31%.Wherein, peptide content is that 80.66%, the 3000 following component of 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 above is only preferred embodiment of the present invention, and is in order to limit the present invention, within the spirit and principles in the present invention not all, any modification of doing, is equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (8)
1. the preparation method of a collagen protein, is characterized in that, comprises the following steps:
(1) pre-treatment: the raw material that will prepare collagen protein cleans up, and then uses 3 ‰-10 ‰ basic solution to soak 10-20min, then raw material is cleaned to neutral, dries 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 ℃ of sterilization 30-40min is cooled to 40 ℃;
(3) add the earthworm serine protease by the 0.5-2 ‰ that takes pretreated raw material weight;
(4) the enzymolysis process condition is controlled: PH is controlled at 6.0-7.8,37-42 ℃ of insulation hydrolysis 6-7h, and 80-85 ℃ of deactivation 30min, cleaner liquid was got in then centrifuging;
(5) using molecular weight cut-off is that 6000 ultrafiltration apparatus carries out ultrafiltration, and adds purified water and make ultrafiltration more complete, gets peritoneal effluent;
(6) peritoneal effluent is carried out drying, obtain collagen protein.
2. the preparation method of collagen protein as described in claim 1, it is characterized in that: in step (1), described basic solution is 5 ‰ NaOH solution.
3. the preparation method of collagen protein as described in claim 1, it is characterized in that: in step (2), sterilising temp is 80 ℃, 30min.
4. the preparation method of collagen protein as described in claim 1, it is characterized in that: in step (3), the amount that adds the earthworm serine protease is to take 1 ‰ of pretreated raw material weight.
5. the preparation method of collagen protein as described in claim 1, it is characterized in that: in step (4), PH is controlled at 6.5, and the temperature of insulation hydrolysis is 40 ℃, hydrolysis time 6.5 hours, the deactivation temperature is 80 ℃.
6. the preparation method of collagen protein as described in claim 1, it 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), and described dry lyophilize or the spraying drying of adopting.
8. the preparation method of collagen protein as described in claim 1, it 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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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017072A (en) * | 2014-06-18 | 2014-09-03 | 常州药物研究所有限公司 | Complete device for collagen purification |
| 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 |
| CN107073076A (en) * | 2014-11-11 | 2017-08-18 | 株式会社日皮 | Immune activation agent, cellular immunity activator and T cell multiplication agent |
| 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 |
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| 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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2013
- 2013-02-28 CN CN201310065180.7A patent/CN103146789B/en active Active
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 |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104017072A (en) * | 2014-06-18 | 2014-09-03 | 常州药物研究所有限公司 | Complete device for collagen purification |
| CN107073076A (en) * | 2014-11-11 | 2017-08-18 | 株式会社日皮 | Immune activation agent, cellular immunity activator and T cell multiplication 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 |
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