CN104535637B - Method for verifying macromolecular collagen or gelatin via pepsase enzymolysis - Google Patents
Method for verifying macromolecular collagen or gelatin via pepsase enzymolysis Download PDFInfo
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- CN104535637B CN104535637B CN201410766878.6A CN201410766878A CN104535637B CN 104535637 B CN104535637 B CN 104535637B CN 201410766878 A CN201410766878 A CN 201410766878A CN 104535637 B CN104535637 B CN 104535637B
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Abstract
The invention discloses a method for verifying macromolecular collagen or gelatin via pepsase enzymolysis. An SDS-PAGE method is used for displaying different peptide segments to obtain an enzymatic fragment atlas, carrying out enzymolysis on collagen with different sources to obtain different peptide segment atlases, and distinguishing the sources of collagen or gelatin according to the peptide segment atlases. The method disclosed by the invention is simple, and the result is accurate.
Description
Technical field
The present invention relates to a kind of method of stomach cardia enzymolysis identification macromolecular collagen protein or gelatin, belongs to biological detection neck
Domain.
Background technology
Gelatin is the product after macromolecular collagen protein is heated after exploded, all has α chains composition in molecular structure
Triple helix conformation (i.e. collagen domain).Collagen present in organism is a kind of white, opaque, unbranched fiber egg
White matter, it is primarily present in the skin of animal, bone, cartilage, tooth, tendon flesh, ligament and blood vessel, is the epochmaking knot of connective tissue
Structure albumen, plays the function of supporting Organoprotective body.And gelatin to be then collagen be heated generation exploded or uncoiling
Product afterwards.At present collagen or gelatin are mainly derived from the tissue such as the fish-skin of pigskin, ox bone or aquatic livestock, fish-bone, extensively
It is general to be applied to the industries such as food, chemical industry, medicine, beauty.The collagen or gelatin of separate sources has different application regions.
Due to due to religion, aquatic livestock collagen has wider application region.But due to collagen and gelatin product
All there is similar triple helix structure, conventional method of protein measurement such as Kjeldahl's method, biuret method, ultraviolet spectrometry light
Degree method, has no idea the source of difference collagen, i.e. the collagen of different plant species is distinguished at present relatively difficult.
The content of the invention
It is an object of the invention to provide a kind of method of stomach cardia enzymolysis identification macromolecular collagen protein or gelatin.
Realize that the object of the invention technical solution is：
A kind of method of stomach cardia enzymolysis identification macromolecular collagen protein or gelatin, its step is as follows：
(1) dissolution of raw material：Macromolecular collagen protein or gelatin 5-50mg are taken, the acetic acid of 1-50 milliliter 0.1-0.5mol is dissolved in
In, it is made into the protein solution that concentration is 0.1-10mg/ml；
(2) heat denatured：Protein solution is heated in 30-100 DEG C of water-bath, the heat time is 10-100 minutes；
(3) pepsin is added：Add pepsin in protein solution again, pepsic consumption is macromolecular glue
The 0.1wt%-10wt% of former albumen or gelatin consumption；
(4) enzyme digestion reaction：To add pepsic protein solution that 10-100 minutes, reaction temperature are reacted in water-bath
For 10-60 DEG C；
(5) enzymolysis terminates：1 milliliter of reacted solution is taken, pepsin inhibitor PMSF (phenylmethylsulfonyl fluoride) is added
5-50 microlitre, terminate enzymolysis process；
(6) electrophoresis Sample is prepared：In the protein solution for terminating enzymolysis process, concentration is added for 0.1-10mol/L's
10-100 microlitre of Tris solution (trishydroxymethylaminomethane), fully mixes and is heated 1-10 minutes in boiling water bath；
(7) electrophoresis Sample storage：Sample after boiling water bath is heated is cooled to immediately 0 DEG C.
(8) electrophoresis：Carry out SDS-PAGE electrophoresis tests.
The present invention principle be：Although the collagen or gelatin of separate sources are respectively provided with similar triple-helix structure,
The amino acid sequence of the α chain subunits of composition triple-helix structure is differed, and through pepsic enzymolysis different eggs can be produced
White matter fragment (peptide fragment).Using the method for SDS-PAGE, different peptide fragments is shown, that is, obtain the collection of illustrative plates of enzymatic fragment.
The collagen of separate sources obtains different peptide fragment collection of illustrative plates after enzymolysis, and according to peptide fragment collection of illustrative plates collagen can be distinguished
Or the source of gelatin.
Compared with prior art, it is an advantage of the invention that：The source of macromolecular collagen protein or gelatin can be reflected
Not, method is simple, result is accurate.
Description of the drawings
Fig. 1 is the peptide fragment collection of illustrative plates Jing after the pepsin of present invention enzymolysis.
1：Macromolecule standard protein label；2：The collagen that silver carp skin is extracted；3：Silver carp skin extracts collagen
Peptide hydrolysis collection of illustrative plates；4：Silver carp squama extracts the peptide hydrolysis collection of illustrative plates of collagen；5：The enzymolysis of the collagen that carp skin is extracted
Peptide fragment collection of illustrative plates.
Specific embodiment
Example 1：
(1) dissolution of raw material：The macromolecular collagen protein 6mg for coming from silver carp skin is fetched, the acetic acid of 24 milliliters of 0.2mol is dissolved in
In, it is made into the protein solution that concentration is 0.25mg/ml.
(2) heat denatured：The macromolecular collagen protein being made into or gelatin solution are heated in 50 DEG C of water-baths, the heat time
For 60 minutes.
(3) protease is added：Pepsin (1 is added in the solution:10000), enzyme preparation is purchased from sigma companies, its
Consumption is the 2% of protein sample consumption.
(4) enzyme digestion reaction：To add pepsic macromolecular collagen protein or gelatin solution that 50 are reacted in water-bath
Minute, reaction temperature is 30 DEG C.
(5) enzymolysis terminates：1 milliliter of reacted solution is taken, PMSF30 microlitre of pepsin inhibitor is added, terminates enzymolysis
Process.
(6) electrophoresis Sample is prepared：In the protein solution of terminating reaction, concentration is added for the Tris solution 50 of 2mol/L
Microlitre, fully mix and heated 6 minutes after boiling water bath.
(7) electrophoresis Sample storage：Sample after boiling water bath is heated is cooled to immediately 0 DEG C, and cooling can be carried out in frozen water,
Or it is put into household freezer.
(8) electrophoresis：SDS-PAGE electrophoresis is carried out, bibliography refers to Laemmli (1970) .Laemmli, U.K.
(1970).Cleavage of structural proteins during assembly head of bacteriophage
T4.Nature, macromolecule protein label used is purchased from Sigma Co., USA, each sample sample in 277,680-685. experiments
Measure as 10 micrograms, experimental result is shown in the 3rd band of Fig. 1, test result indicate that through the method, can be to macromolecular glue
The source of former albumen or gelatin is differentiated.
Example 2：
(1) dissolution of raw material：Silver carp squama macromolecular collagen protein 8mg is taken, in being dissolved in the acetic acid of 16 milliliters of 0.3mol, is made into dense
Spend the protein solution for 0.5mg/ml.
(2) heat denatured：The macromolecular collagen protein solution being made into is heated in 55 DEG C of water-baths, the heat time is 70 points
Clock.
(3) protease is added：Pepsin (1 is added in the solution:10000), enzyme preparation is purchased from sigma companies, stomach
The consumption of protease is the 6% of protein sample consumption
(4) enzyme digestion reaction：To add pepsic macromolecular collagen protein or gelatin solution that 70 are reacted in water-bath
Minute, reaction temperature is 30 DEG C.
(5) enzymolysis terminates：1 milliliter of reacted solution is taken, adds 10 microlitres of pepsin inhibitor, termination to digest
Journey.
(6) electrophoresis Sample is prepared：In the protein solution of terminating reaction, concentration is added for the Tris solution 30 of 5mol/L
Microlitre, fully mix and heated 5 minutes after boiling water bath.
(7) electrophoresis Sample storage：Sample after boiling water bath is heated is cooled down immediately, and cooling can be carried out in frozen water,
Or it is put into household freezer.
(8) electrophoresis：SDS-PAGE electrophoresis is carried out, bibliography refers to Laemmli (1970) .Laemmli, U.K.
(1970).Cleavage of structural proteins during assembly head of bacteriophage
T4.Nature, macromolecule protein label used is purchased from Sigma Co., USA, each sample spot in 277,680-685. experiments
Sample amount is 10 micrograms, and experimental result is shown in the 4th band of Fig. 1, test result indicate that through the method, can be to macromolecular
The source of collagen or gelatin is differentiated.
Example 3：
(1) dissolution of raw material：Carp skin macromolecular collagen protein 10mg is taken, in being dissolved in the acetic acid of 50 milliliters of 0.5mol, is made into
Concentration is the protein solution of 0.2mg/ml.
(2) heat denatured：The macromolecular collagen protein being made into or gelatin solution are heated in 60 DEG C of water-baths, the heat time
For 50 minutes.
(3) protease is added：Pepsin (1 is added in the solution:10000), enzyme preparation is purchased from sigma companies, stomach
The consumption of protease is the 4% of protein sample consumption.
(4) enzyme digestion reaction：To add pepsic macromolecular collagen protein or gelatin solution that 60 are reacted in water-bath
Minute, reaction temperature is 20 DEG C.
(5) enzymolysis terminates：1 milliliter of reacted solution is taken, adds 40 microlitres of pepsin inhibitor, termination to digest
Journey.
(6) electrophoresis Sample is prepared：In the protein solution of terminating reaction, concentration is added for the Tris solution 60 of 8mol/L
Microlitre, fully mix and heated 4 minutes after boiling water bath.
(7) electrophoresis Sample storage：Sample after boiling water bath is heated is cooled down immediately, and cooling can be carried out in frozen water,
Or it is put into household freezer.
(8) electrophoresis：SDS-PAGE electrophoresis is carried out, bibliography refers to Laemmli (1970) .Laemmli, U.K.
(1970).Cleavage of structural proteins during assembly head of bacteriophage
T4.Nature, macromolecule protein label used is purchased from Sigma Co., USA, each sample spot in 277,680-685. experiments
Sample amount is 10 micrograms, and experimental result is shown in the 5th band of Fig. 1, test result indicate that through the method, can be to macromolecular
The source of collagen or gelatin is differentiated.
Claims (3)
1. a kind of method that stomach cardia enzymolysis identifies macromolecular collagen protein or gelatin, it is characterised in that step is as follows：
（1）Dissolution of raw material：Take macromolecular collagen protein or gelatin is dissolved in the acetic acid of 0.1-0.5mol, be made into concentration for 0.1-
The protein solution of 10mg/ml；
（2）Heat denatured：Protein solution is heated in 30-100 DEG C of water-bath；
（3）Add pepsin：Add pepsin in protein solution again, pepsic consumption is macromolecular collagen egg
The 0.1wt%-10wt% of white or gelatin consumption；
（4）Enzyme digestion reaction：Pepsic protein solution will be added to react 10-100 minutes in water-bath, reaction temperature is
10-60℃；
（5）Enzymolysis terminates：1 milliliter of reacted solution is taken, 5-50 microlitre of pepsin inhibitor is added, terminates enzymolysis process；
（6）Prepare electrophoresis Sample：In the protein solution for terminating enzymolysis process, concentration is added for three hydroxyls of 0.1-10mol/L
10-100 microlitre of aminomethane solution, fully mixes and is heated 1-10 minutes in boiling water bath；
（7）Electrophoresis Sample is stored：Sample after boiling water bath is heated is cooled to immediately 0 DEG C；
（8）Electrophoresis：Carry out SDS-PAGE electrophoresis tests.
2. the method that stomach cardia enzymolysis as described in claim 1 identifies macromolecular collagen protein or gelatin, it is characterised in that
Step（2）In heat time be 10-100 minutes.
3. the method that stomach cardia enzymolysis as described in claim 1 identifies macromolecular collagen protein or gelatin, it is characterised in that
Step（5）In pepsin inhibitor adopt phenylmethylsulfonyl fluoride.
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Cited By (1)
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| RU2816712C1 (en) * | 2023-09-12 | 2024-04-03 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Кемеровский государственный университет" | Method of producing gelatine |
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| CN104985051B (en) * | 2015-06-09 | 2017-04-12 | 苏州昀冢电子科技有限公司 | Machining method of mobile terminal card holder |
| CN107727861B (en) * | 2017-08-22 | 2019-10-22 | 厦门依柯利斯医疗科技有限公司 | A kind of pepsin assay kit and measuring method |
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| JP3502544B2 (en) * | 1998-06-05 | 2004-03-02 | 株式会社ニッピ | New gelatin and its manufacturing method |
| CA2243230A1 (en) * | 1998-07-15 | 2000-01-15 | Aled Edwards | A device and method for the determination of protein domain boundaries |
| JP2007282515A (en) * | 2006-04-12 | 2007-11-01 | Applied Cell Biotechnologies Inc | Method for producing collagens and collagens |
| CN101858885A (en) * | 2009-04-08 | 2010-10-13 | 黄玲惠 | Method of quantitative analysis of collagen by means of capillary electrophoresis and set reagent |
| CN102621213A (en) * | 2012-01-13 | 2012-08-01 | 淮海工学院 | Evaluation method for degree of thermal denaturation of macromolecular collagen by using pepsin process |
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