CN104073537A - Preparation method for active collagen from compound protease - Google Patents
Preparation method for active collagen from compound protease Download PDFInfo
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- CN104073537A CN104073537A CN201310096815.XA CN201310096815A CN104073537A CN 104073537 A CN104073537 A CN 104073537A CN 201310096815 A CN201310096815 A CN 201310096815A CN 104073537 A CN104073537 A CN 104073537A
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Abstract
The invention discloses a preparation method for active collagen from compound protease. The method comprises the following steps: 1) adding compound protease into pretreated fish skin mixed liquor for enzymatic hydrolysis, wherein the compound protease at least comprises two selected from the group consisting of papain, bromelain, trypsin, neutral protease, animal protein hydrolase, acidic protease and pepsin; 2) subjecting the obtained enzymatic hydrolysis liquid to centrifugation and taking supernatant; 3) subjecting the supernatant to ultrafiltration and concentration and then to salting-out; and 4) carrying out ultrafiltration, desalination and concentration; wherein the fish skin mixed liquor has a concentration of 2.0 to 8.0 wt% and a pH value of 2 to 6, and the addition amount of the compound protease is 2.0 to 8.0 wt% of the weight of the fish skin mixed liquor. The method provided by the invention has high security and no side-effects; and the prepared active collagen has the advantages of a high extraction rate, high purity, high thermal denaturation temperature and the like.
Description
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Technical field
The invention belongs to biochemical field, relate in particular to the extracting method of collagen protein.
Background technology
Domestic at present at the early-stage to utilizing prozyme to extract the research of active collagen, domestic Shuo Suo colleges and universities have carried out the research that prozyme extracts active collagen, at aspects such as the selection of proteolytic enzyme, enzymolysis process, obtain certain progress.Adopt at present enzymolysis process to extract active collagen, the proteolytic ferment of application is comparatively single, mainly contains trypsinase, stomach en-, Sumizyme MP, neutral protease, papoid etc.For the biological activity of most of active collagens, its suitability for industrialized production also has certain difficulty, is mainly that separation and purification difficulty are large because of enzyme easily sex change in collagen hydrolysate process, and detection means and technology are not mature enough.
Summary of the invention
Goal of the invention: for the problem and shortage of above-mentioned existing existence, the object of this invention is to provide a kind of method of utilizing compound protease to prepare active collagen.
Technical scheme: for achieving the above object, the present invention by the following technical solutions: a kind of method of utilizing compound protease to prepare active collagen, comprises the following steps: 1) add compound protease to carry out enzymolysis through pretreated fish-skin mixed solution; Wherein said compound protease at least comprises in papoid, bromeline, trypsinase, neutral protease, animal proteolytic enzyme, aspartic protease and stomach en-two kinds; 2) enzymolysis solution obtaining is carried out to centrifugal treating, get supernatant liquor; 3) will after supernatant liquor ultrafiltration and concentration, saltout; 4) again carry out ultrafiltration desalination concentrated; Wherein, the concentration of fish-skin mixed solution is 2.0～8.0wt%, and pH is 2～6, and the addition of described compound protease is 2.0～8.0wt% of fish-skin mixed solution weight.
As preferably, the hydrolysis temperature of step 1) is 4～16 ℃, and the time is 6～10h, and during reaction, the pH of system is controlled at 5.0～9.0.
As preferably, in step 1), also add inhibitor.
As preferably, step 2) in, centrifugal rotational speed is 4000～5000r/min.
As preferably, the working pressure of step 3) ultrafiltration and concentration is 0.15-0.35MPa, temperature 4-10 ℃, and pH 2-6, ultra-filtration membrane aperture is 10-50nm.
As preferably, in step 3), to saltout while processing, collagen solution concentration is 2.5-5.5%, and NaCl concentration is 10-30%, and pH value is 2-6, and temperature is 4-10 ℃.
As preferably, the operational condition of step 4) ultrafiltration and concentration is as follows: working pressure 0.5-1.0Mpa, and temperature is 4-10 ℃, pH2-6, ultra-filtration membrane aperture is 100-200nm.
Beneficial effect: compared with prior art, the present invention has the following advantages: the present invention utilizes single factor experiment and orthogonal test to be optimized the hydrolysising condition of compound protease, set up pH, hydrolysis temperature, enzymolysis time, the ratio of enzyme-to-substrate, the relation of concentration of substrate and enzymic hydrolysis degree, the optimum hydrolysising condition that obtains compound protease is: trypsinase and papoid ratio are 1-3:1, the consumption of compound protease is the 2.0-8.0wt% of fish-skin mixed solution quality, concentration of substrate 2.0-8.0wt%, pH5.0-9.0, enzymolysis 6-10h under 4-16 ℃ of condition, its the highest collagen protein extraction rate reached to 91.2%.
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention, should understand these embodiment is only not used in and limits the scope of the invention for the present invention is described, after having read the present invention, those skilled in the art all fall within the application's claims limited range to the modification of the various equivalent form of values of the present invention.
A kind of method of utilizing compound protease to prepare active collagen, by in 2.0～8.0% fish-skin mixed solution, regulate pH to 2～6 to carry out pre-treatment, then add papoid, trypsinase and pepsic compound protease, add-on is identical with fish-skin content, and controlling temperature at 10～12 ℃, enzymolysis 8h controls pH between 5～9 in this process.Then the supernatant liquor after enzymolysis is carried out to centrifugal treating, rotating speed is in 4500r/min left and right.
Solution after centrifugal is carried out to ultrafiltration and concentration for the first time, and working pressure is 0.15-0.35MPa, temperature 4-10 ℃, and pH 2-6, ultra-filtration membrane aperture is 10-50nm; Then saltout: collagen solution concentration is 2.5-5.5%, NaCl concentration is 10-30%, and pH value is 2-6, and temperature is 4-10 ℃; Finally carry out ultrafiltration for the second time: working pressure 0.5-1.0Mpa, temperature is 4-10 ℃, pH2-6, ultra-filtration membrane aperture is 100-200nm.
The present invention utilizes single factor experiment and orthogonal test to be optimized the hydrolysising condition of compound protease, set up the relation of ratio, concentration of substrate and the enzymic hydrolysis degree of pH, hydrolysis temperature, enzymolysis time, enzyme-to-substrate, the optimum hydrolysising condition that obtains compound protease is: trypsinase and papoid ratio are 1-3:1, the consumption of compound protease is the 2.0-8.0wt% of fish-skin mixed solution quality, concentration of substrate 2.0-8.0wt%, pH5.0-9.0, enzymolysis 6-10h under 4-16 ℃ of condition, its highest collagen protein extraction rate reached to 91.2%.
On this basis, the present invention again system optimization and screened ultrafiltration and concentration, saltoutd, dialysis desalting, ultrafiltration remove the processing condition such as small molecules, match with the hydrolysising condition of compound protease above, set up the method for a set of complete conjugated protein enzyme extraction active collagen.
The inventive method is safe, has no side effect, and preparation-obtained active collagen has the advantages such as extraction yield, purity and thermal denaturation temperature.
Claims (7)
1. utilize compound protease to prepare a method for active collagen, comprise the following steps:
1) through pretreated fish-skin mixed solution, add compound protease to carry out enzymolysis; Wherein said compound protease at least comprises in papoid, bromeline, trypsinase, neutral protease, animal proteolytic enzyme, aspartic protease and stomach en-two kinds;
2) enzymolysis solution obtaining is carried out to centrifugal treating, get supernatant liquor;
3) will after supernatant liquor ultrafiltration and concentration, saltout;
4) again carry out ultrafiltration desalination concentrated;
Wherein, the concentration of fish-skin mixed solution is 2.0～8.0wt%, and pH is 2～6, and the addition of described compound protease is 2.0～8.0wt% of fish-skin mixed solution weight.
2. utilize according to claim 1 compound protease to prepare the method for active collagen, it is characterized in that: the hydrolysis temperature of step 1) is 4～16 ℃, and the time is 6～10h, and during reaction, the pH of system is controlled at 5.0～9.0.
3. utilize according to claim 2 compound protease to prepare the method for active collagen, it is characterized in that: in step 1), also add inhibitor.
4. utilize according to claim 2 compound protease to prepare the method for active collagen, it is characterized in that: step 2) in centrifugal rotational speed be 4000～5000r/min.
5. utilize according to claim 2 compound protease to prepare the method for active collagen, it is characterized in that: the working pressure of step 3) ultrafiltration and concentration is 0.15-0.35MPa, temperature 4-10 ℃, pH 2-6, ultra-filtration membrane aperture is 10-50nm.
6. utilize according to claim 2 compound protease to prepare the method for active collagen, it is characterized in that: in step 3), saltout while processing, collagen solution concentration is 2.5-5.5%, and NaCl concentration is 10-30%, and pH value is 2-6, and temperature is 4-10 ℃.
7. utilize according to claim 2 compound protease to prepare the method for active collagen, it is characterized in that: the operational condition of step 4) ultrafiltration and concentration is as follows: working pressure 0.5-1.0Mpa, temperature is 4-10 ℃, pH2-6, and ultra-filtration membrane aperture is 100-200nm.
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| CN201310096815.XA CN104073537A (en) | 2013-03-25 | 2013-03-25 | Preparation method for active collagen from compound protease |
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| CN201310096815.XA CN104073537A (en) | 2013-03-25 | 2013-03-25 | Preparation method for active collagen from compound protease |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105995653A (en) * | 2016-05-13 | 2016-10-12 | 辽宁石油化工大学 | Method for preparing artemia-cysts-shell hydrolyzing liquid |
| CN107227331A (en) * | 2017-07-11 | 2017-10-03 | 浙江丰宇海洋生物制品有限公司 | A kind of method of the molten slurry production protein small peptide of utilization fish |
| CN107576737A (en) * | 2017-09-05 | 2018-01-12 | 四川农业大学 | A kind of method based on fish scale collagen tunica fibrosa detection glyphosate |
| CN111808215A (en) * | 2020-07-10 | 2020-10-23 | 上海林清轩生物科技有限公司 | Method for preparing bioactive substances from eggshell membrane by complex enzyme hydrolysis method |
| CN112062834A (en) * | 2020-11-16 | 2020-12-11 | 江西中医药大学 | Deep sea fish skin collagen peptide and extraction and preparation method thereof |
| CN113136412A (en) * | 2021-04-22 | 2021-07-20 | 浙江珂瑞康生物医疗科技有限公司 | Biological load determination treatment liquid for biomedical material containing soluble protein |
| CN113528604A (en) * | 2021-07-13 | 2021-10-22 | 镇江市天益生物科技有限公司 | Fish gelatin hydrolysate with oral hypoglycemic activity and application thereof |
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2013
- 2013-03-25 CN CN201310096815.XA patent/CN104073537A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105995653A (en) * | 2016-05-13 | 2016-10-12 | 辽宁石油化工大学 | Method for preparing artemia-cysts-shell hydrolyzing liquid |
| CN107227331A (en) * | 2017-07-11 | 2017-10-03 | 浙江丰宇海洋生物制品有限公司 | A kind of method of the molten slurry production protein small peptide of utilization fish |
| CN107576737A (en) * | 2017-09-05 | 2018-01-12 | 四川农业大学 | A kind of method based on fish scale collagen tunica fibrosa detection glyphosate |
| CN111808215A (en) * | 2020-07-10 | 2020-10-23 | 上海林清轩生物科技有限公司 | Method for preparing bioactive substances from eggshell membrane by complex enzyme hydrolysis method |
| CN111808215B (en) * | 2020-07-10 | 2022-05-27 | 上海林清轩生物科技有限公司 | Method for preparing bioactive substances from eggshell membrane by complex enzyme hydrolysis method |
| CN112062834A (en) * | 2020-11-16 | 2020-12-11 | 江西中医药大学 | Deep sea fish skin collagen peptide and extraction and preparation method thereof |
| CN113136412A (en) * | 2021-04-22 | 2021-07-20 | 浙江珂瑞康生物医疗科技有限公司 | Biological load determination treatment liquid for biomedical material containing soluble protein |
| CN113528604A (en) * | 2021-07-13 | 2021-10-22 | 镇江市天益生物科技有限公司 | Fish gelatin hydrolysate with oral hypoglycemic activity and application thereof |
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Application publication date: 20141001 |