CN102621213A - Evaluation method for degree of thermal denaturation of macromolecular collagen by using pepsin process - Google Patents
Evaluation method for degree of thermal denaturation of macromolecular collagen by using pepsin process Download PDFInfo
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- CN102621213A CN102621213A CN2012100096842A CN201210009684A CN102621213A CN 102621213 A CN102621213 A CN 102621213A CN 2012100096842 A CN2012100096842 A CN 2012100096842A CN 201210009684 A CN201210009684 A CN 201210009684A CN 102621213 A CN102621213 A CN 102621213A
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Abstract
The invention discloses an evaluation method for the degree of thermal denaturation of macromolecular collagen by using a pepsin process. The method is characterized by comprising the following steps: (1) selection of a raw material: a step of selecting leftovers of aquatic product processing-fishskin as the raw material; (2) immersion and dissolving: a step of adding acetic acid with a concentration of 0.01 to 1.0 mol/L into the raw material to obtain a collagen solution; (3) heat pretreatment: a step of heating the collagen solution, shaking the solution every five minutes during heating and carrying out rapid cooling after heating is finished; (4) enzymatic hydrolysis: a step of adding pepsin into the collagen solution, carrying out a heat reaction and adding phenylmethylsulfonyl fluoride after the heat reaction is finished so as to terminate the reaction; and (5) preparation of an electrophoresis sample and electrophoresis. The invention has the following advantages: used equipment is simple; more comprehensive and visual reflection of the denaturation state of protein is realized.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of evaluation method of stomach cardia enzyme process macromolecular collagen protein thermal denaturation degree.
Background technology
Macromolecular collagen protein is meant that collagen molecules has complete triple-helix structure, and molecular weight is at the collagen more than 300,000.Former studies collagen thermal denaturation degree methods mainly contains viscosimetry, DSC method, circular dichroism spectrometry (CD method) etc.Viscosimetry is according under the heating state, and the principle that collagen molecules uncoiling, fracture cause solution viscosity to descend is measured; The DSC method can be followed the process of thermal absorption based on triple helical disintegration process; The CD method is measured based on the α spiral that exists in the collagen, has in certain wavelengths just to absorb or the principle of negative absorption.No matter but which kind of method all will have corresponding apparatus, particularly CD method, DSC method are had relatively high expectations for equipment, and the mensuration result receives than multifactor impact.The equipment that the thermal denaturation degree that adopts enzyme process (pepsin, chymotrypsin etc.) to estimate collagen not only adopts is simple; And both can reflect the albumen situation that has complete triple-helix structure in the collagen through electrophoretogram; The situation that can reflect the fragment after the dimer, monomer and the subunit that are made up of the collagen subunit decompose again is comprehensive more and directly perceived to the reflection of protein denaturation situation.
Summary of the invention
The present invention provides a kind of pepsin that adopts to combine the SDS-PAGE method to estimate the thermal denaturation degree of collagen; It has but the equipment that adopts is simple; And both can reflect the albumen situation that has complete triple-helix structure in the collagen through electrophoretogram; The situation that can reflect the fragment after the dimer that is made up of the collagen subunit, monomer and subunit decompose again, to the reflection of protein denaturation situation more comprehensively with advantage intuitively.
For achieving the above object, the present invention adopts following technical scheme:
A kind of evaluation method of stomach cardia enzyme process macromolecular collagen protein thermal denaturation degree, step is following:
(1) material choice, selecting the leftover bits and pieces-fish-skin of processing of aquatic products is raw material, also can select other animal tissues of being rich in collagen such as pigskin, ox-hide, muscle tendon.
(2) soak dissolving: raw material adds 0.01-1.0mol/L acetic acid in step (1), obtains collagen solution, and the concentration of collagen solution is 0.1-20%.
(3) heating pretreatment: with the collagen solution heating that obtains in the step (2), be 10-200 minute heat time heating time, and heating-up temperature is 20 ℃-80 ℃, in the heating process, whenever rocks once at a distance from 5 minutes, cooling cooling rapidly after heating is accomplished.
(4) enzymolysis: add pepsin in the collagen liquid that in step (3), obtains, addition is: 1-20%, time 10-100 minute, adds the phenylmethylsulfonyl fluoride cessation reaction after reaction is accomplished by temperature of reaction 10-60 ℃.
(5) make electrophoresis sample and electrophoresis: the collagen solution of cessation reaction is added sodium dodecyl sulfate solution making electrophoresis sample and electrophoresis.
Preferably, the acetate concentration that adds in the step (2) is 0.04-0.8mol/L, and the concentration of collagen solution is 5-15%.
Preferably, be 20-180 minute heat time heating time in the step (3), and heating-up temperature is 20 ℃-80 ℃.
Preferably, in the step (4), adding pepsic amount during enzymolysis is 5-15%.
Preferably, in the step (4), heating-up temperature is 15-55 ℃, and be 20-50 minute heat time heating time.
Preferably, the thermal denaturation degree of collagen is represented according to the reduction of β chain concentration in the collagen in the step (5), and the degree of the former protein thermal denaturation of gelatin of speaking more more that β chain concentration reduces is big more.
Advantage of the present invention:
The equipment that adopts is simple; And both can reflect the albumen situation that has complete triple-helix structure in the collagen through electrophoretogram; The situation that can reflect the fragment after the dimer, monomer and the subunit that are made up of the collagen subunit decompose again is comprehensive more and directly perceived to the reflection of protein denaturation situation.
Description of drawings
Fig. 1 is a process flow diagram of the present invention.
Fig. 2 is the electrophoresis pattern behind the different temperatures enzymolysis, denaturation temperature enzymolysis 1/10 1:28 ℃, and 2:30 ℃, 3:31.5 ℃, 4:33 ℃, 5:35 ℃, 6:40 ℃, 7:50 ℃.
Embodiment
(1) material choice, selecting the leftover bits and pieces-fish-skin of processing of aquatic products is raw material, also can select other animal tissues of being rich in collagen such as pigskin, ox-hide, muscle tendon.
(2) soak dissolving: raw material adds 0.01-1.0mol/L acetic acid in step (1), obtains collagen solution, and the concentration of collagen solution is 0.1-20%.
(3) heating pretreatment: with the collagen solution heating that obtains in the step (2), be 10-200 minute heat time heating time, and heating-up temperature is 20 ℃-80 ℃, in the heating process, whenever rocks once at a distance from 5 minutes, cooling cooling rapidly after heating is accomplished.
(4) enzymolysis: add pepsin in the collagen liquid that in step (3), obtains, addition is: 1-20%, time 10-100 minute, adds the phenylmethylsulfonyl fluoride cessation reaction after reaction is accomplished by temperature of reaction 10-60 ℃.
(5) make electrophoresis sample and electrophoresis: the collagen solution of cessation reaction is added sodium dodecyl sulfate solution making electrophoresis sample and electrophoresis.
Embodiment
As shown in Figure 2; Get ox-hide, carp skin, cod collagen sample; Be mixed with 0.5% 0.5M acetum, and heating added 1/10 pepsin after 30 minutes under 1:28 ℃, 2:30 ℃, 3:31.5 ℃, 4:33 ℃, 5:35 ℃, 6:40 ℃, 7:50 ℃ temperature; 20 ℃ the reaction 3 hours after, electrophoresis and analytical electrophoresis collection of illustrative plates.
The above; Be merely embodiment of the present invention, but protection scope of the present invention is not limited thereto, any technician who is familiar with the present technique field is in the technical scope that the present invention discloses; Can expect easily changing or replacement, all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain of said claim.
Claims (6)
1. the evaluation method of a stomach cardia enzyme process macromolecular collagen protein thermal denaturation degree is characterized in that step is following:
(1) material choice, selecting the leftover bits and pieces-fish-skin of processing of aquatic products is raw material, also can select other animal tissues of being rich in collagen such as pigskin, ox-hide, muscle tendon.
(2) soak dissolving: raw material adds 0.01-1.0mol/L acetic acid in step (1), obtains collagen solution, and the concentration of collagen solution is 0.1-20%.
(3) heating pretreatment: with the collagen solution heating that obtains in the step (2), be 10-200 minute heat time heating time, and heating-up temperature is 20 ℃-80 ℃, in the heating process, whenever rocks once at a distance from 5 minutes, cooling cooling rapidly after heating is accomplished.
(4) enzymolysis: add pepsin in the collagen liquid that in step (3), obtains, addition is: 1-20%, time 10-100 minute, adds the phenylmethylsulfonyl fluoride cessation reaction after reaction is accomplished by temperature of reaction 10-60 ℃.
(5) make electrophoresis sample and electrophoresis: the collagen solution of cessation reaction is added sodium dodecyl sulfate solution making electrophoresis sample and electrophoresis.
2. the evaluation method of stomach cardia enzyme process macromolecular collagen protein thermal denaturation degree according to claim 1 is characterized in that: the acetate concentration that adds in the step (2) is 0.04-0.8mol/L, and the concentration of collagen solution is 5-15%.
3. the evaluation method of stomach cardia enzyme process macromolecular collagen protein thermal denaturation degree according to claim 1 is characterized in that: be 20-180 minute heat time heating time in the step (3), and heating-up temperature is 20 ℃-80 ℃.
4. the evaluation method of stomach cardia enzyme process macromolecular collagen protein thermal denaturation degree according to claim 1 is characterized in that: in the step (4), adding pepsic amount during enzymolysis is 5-15%.
5. the evaluation method of stomach cardia enzyme process macromolecular collagen protein thermal denaturation degree according to claim 1 is characterized in that: in the step (4), heating-up temperature is 15-55 ℃, and be 20-50 minute heat time heating time.
6. the evaluation method of stomach cardia enzyme process macromolecular collagen protein thermal denaturation degree according to claim 1 is characterized in that: the denaturation degrees of judging collagen in the step (5) through the variation of collagen β chain.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104535637A (en) * | 2014-12-11 | 2015-04-22 | 淮海工学院 | Method for verifying macromolecular collagen or gelatin via pepsase enzymolysis |
| CN105985429A (en) * | 2015-02-09 | 2016-10-05 | 广州创尔生物技术股份有限公司 | Aseptic collagen liquid with biological activity and preparation method thereof |
| CN107014854A (en) * | 2017-03-29 | 2017-08-04 | 淄博黄河龙生物工程有限公司 | The durothermic detection method of collagen |
| CN107488696A (en) * | 2017-09-19 | 2017-12-19 | 广东雅道生物科技有限公司 | One breeder bone double enzymolysis method |
| CN109884153A (en) * | 2019-03-11 | 2019-06-14 | 淮海工学院 | A kind of judgement new method of I-type collagen triple-helix structure integrality |
| CN110095421A (en) * | 2019-05-17 | 2019-08-06 | 淮海工学院 | A kind of measuring method of collagen denaturation temperature |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101628938A (en) * | 2009-08-12 | 2010-01-20 | 江苏省海洋资源开发研究院 | Extraction technology of macromolecular collagen protein |
| CN101628937A (en) * | 2009-08-12 | 2010-01-20 | 江苏省海洋资源开发研究院 | Purification technology of macromolecular collagen protein |
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| CN101628938A (en) * | 2009-08-12 | 2010-01-20 | 江苏省海洋资源开发研究院 | Extraction technology of macromolecular collagen protein |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104535637A (en) * | 2014-12-11 | 2015-04-22 | 淮海工学院 | Method for verifying macromolecular collagen or gelatin via pepsase enzymolysis |
| CN105985429A (en) * | 2015-02-09 | 2016-10-05 | 广州创尔生物技术股份有限公司 | Aseptic collagen liquid with biological activity and preparation method thereof |
| CN107014854A (en) * | 2017-03-29 | 2017-08-04 | 淄博黄河龙生物工程有限公司 | The durothermic detection method of collagen |
| CN107488696A (en) * | 2017-09-19 | 2017-12-19 | 广东雅道生物科技有限公司 | One breeder bone double enzymolysis method |
| CN107488696B (en) * | 2017-09-19 | 2021-05-07 | 广东雅道生物科技有限公司 | Chicken bone double-enzymolysis method |
| CN109884153A (en) * | 2019-03-11 | 2019-06-14 | 淮海工学院 | A kind of judgement new method of I-type collagen triple-helix structure integrality |
| CN109884153B (en) * | 2019-03-11 | 2021-07-20 | 淮海工学院 | Novel method for judging integrity of triple-helix structure of type I collagen |
| CN110095421A (en) * | 2019-05-17 | 2019-08-06 | 淮海工学院 | A kind of measuring method of collagen denaturation temperature |
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Application publication date: 20120801 |