CN101246140B - Novel method for fast measuring collagen hydrolysis degree by biologic sensor - Google Patents
Novel method for fast measuring collagen hydrolysis degree by biologic sensor Download PDFInfo
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- CN101246140B CN101246140B CN2007100149618A CN200710014961A CN101246140B CN 101246140 B CN101246140 B CN 101246140B CN 2007100149618 A CN2007100149618 A CN 2007100149618A CN 200710014961 A CN200710014961 A CN 200710014961A CN 101246140 B CN101246140 B CN 101246140B
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Abstract
The invention provides a method when alkaline protease hydrolyses from big molecular collagen into minor polypeptides, free amino acids in enzymatic hydrolysate increase gradually, a positive correlation is shown between increased amount and hydrolysis degree, so the hydrolysis degree can be calculated by detecting total amino acids or a certain kind of amino acid content (such as glutamate) in enzymolysis liquid. The invention uses glutamate oxidase biosensor, according to the theory of acid ketone H2O2 reacted from glutamate and glutamate oxidase, the reacted H2O2 contact with H2O2 electrode in biosensor in order to produce current signal, the current signal has a linear relation with glutamate concentration, through computer processing, the result is displayed directly. The method has characteristics of fast speed, accurateness, which can detect and control the process of producing active peptide from collagen in industry.
Description
Technical field:
The present invention relates to the assay method of collagen hydrolysate degree, belong to the Applied Biotechnology field.Be specifically related to use the method for dglutamic oxidase biosensor assay collagen hydrolysate degree.Adopt the inventive method to measure degree of hydrolysis, accurately, fast, can in 30s, draw data; And can be online with microcomputer; Implement printing, data analysis and control function automatically, can in common laboratory is analyzed, use, also can application in the industrialization that the collagen enzymolysis prepares active peptide is produced.
Background technology:
The collagen that the aquatic livestock body contains has identical characteristics with the collagen that the Lu Sheng mammalian body contains, and all is the triple helix structure that is made up of three polypeptied chains, and amino acid whose main composition also is glycocoll, proline and hydroxyproline.Simultaneously, the collagen in the aquatic livestock body also has some self characteristics and effect.
Compare with land animal, the aquatic product collagen has the characteristics of himself.The proline in the isinglass and the content of hydroxyproline are more much lower than animal gelatin, and the content of the methionine in the isinglass is more much higher than animal gelatin.Because the proline in the isinglass and the content of hydroxyproline are lower than terrestrial animal gelatin; Protein structure is easy to destroy in process of production; Cause in the component of its albumen and have the γ component hardly; And in low-molecular-weight α component concentration more, thereby the gelation temperature of isinglass is lower than terrestrial animal gelatin.
Containing multiple biologically active peptide in the collagen, as suppressing hypertensin conversion enzyme activity, suppressing blood platelet congealing activity, antioxidation activity, antitumor activity etc.Collagen active peptide is to be raw material with collagen, and through the directed gradient hydrolyzed product of multiple protein enzyme, its molecular weight is below 2000Da.The mode of production of active peptide mainly contains three kinds at present: extract from the natural biological body (1); (2) the vitro enzyme aminosal produces; (3) synthetic through chemical method and recombinant DNA technology.The different production mode respectively has characteristics, and this depends on length, quantity and the purposes of purpose peptide.The content of biologically active peptide seldom can be used as biologically active peptide research in the natural products, is infeasible and be used as food.Chemical synthesis is widely used in the pharmaceutical grade peptide of producing high price, but its cost is high, and accessory substance can be harmful.The recombinant DNA technology synthetic method also is widely used, but this method is used for producing long peptide and protein more, and many biologically active peptides all are small peptides, so also limited the use of this method.The Production by Enzymes biologically active peptide has lot of advantages, i.e. gentle, safe, the inexpensive and control of working condition just can obtain specific active peptide in the reaction time, and present enzyme process is used widely in biologically active peptide production.On the proteolysis Products Development, the exploitation of peptide possibly already started the chance that makes new advances for traditional food.
Though the Production by Enzymes biologically active peptide has lot of advantages; But when enzymolysis prepares active peptide in industry; Still there is complicated, loaded down with trivial details, the restive problem of enzymolysis process; Need to inquire into a kind of succinctly, fast, the assay method that can be used for production control is with the control enzymolysis process, thereby realizes the large-scale production that enzymolysis prepares active peptide.
Summary of the invention:
The objective of the invention is to exist when preparing active peptide enzymolysis process complicated, loaded down with trivial details, restive problem proposition, the present invention to can be the industrialization production that the collagen enzymolysis prepares active peptide a kind of new method of fast, accurately measuring the enzymolysis liquid degree of hydrolysis is provided to enzymolysis.
Step 1: the preparation of alkali protease enzymolysis liquid.
45 ℃ of optimum hydrolysis temperature, pH8.5, under the enzyme concentration 90mg/kg condition, the alkali protease enzymolysis liquid that the preparation concentration of substrate is 5%, enzymolysis time is respectively 1.5h, 2.0h, 2.5h, 3.0h, 3.5h, 4.0h, 4.5h.
Step 2: the mensuration of alkali protease enzymolysis liquid glutamic and degree of hydrolysis.
Through the above-mentioned enzymolysis liquid degree of hydrolysis size of potentiometric determination.Draw enzymolysis liquid 5ml in the 100ml volumetric flask, constant volume.Draw 20ml in the 200ml beaker, add 60ml distilled water, start magnetic stirring apparatus, indicate pH8.2 with 0.05mol/L NaOH standard solution titration to acidometer, record consumes the ml number of NaOH; The neutral formalin that adds 10ml40% again, mixing.Continue titration to pH9.2 with above-mentioned NaOH standard solution, record consumes the ml number of NaOH standard solution, does blank assay simultaneously.Mensuration result is as shown in Figure 1.With the prolongation of enzymolysis time, the cod collagen degree of hydrolysis is increase tendency gradually.
Glutamic is measured and is adopted SBA-40C type biosensor analysis.Start is cleaned once automatically.After green light is bright, when screen is in 0 value of auto zero state, inject injection port drawing 50 good μ L standard models.After calibration is accomplished, add 50 μ L enzymolysis liquids and carry out glutamic mensuration.Mensuration result is as shown in Figure 2.With the prolongation of enzymolysis time, glutamic also is increase tendency gradually in the cod collagen enzymolysis liquid.
Step 3: the regretional analysis of glutamic and degree of hydrolysis size.
As can beappreciated from fig. 3, data point drops near the straight line basically.Find out that thus there is linear relationship in variable X (glutamic) with the relation of Y (degree of hydrolysis).But owing to be not that all data points drop on the straight line fully, so the relation of X and Y is indefinite in the degree that can be confirmed a Y value uniquely by an X value, promptly exists deviation between actual measured value and the regressand value.According to experimental data, obtain regression equation y=0.7811x+5.9172, though be through related coefficient check (| r|=0.9658＞r
Min=0.754), F check (F=69.46＞F
0.01(1; 5) still be standard error (0.251) (seeing table 1, table 2)=16.26); Show that all X (glutamic) and Y (degree of hydrolysis) have very significant linear relationship, prove through measuring glutamic and predict that tested zymolyte degree of hydrolysis size has actual application value.
Table 1 regression statistical analysis
| df | SS | MS | F | F 0.01(1，5) | Conspicuousness | |
| |
1 | 4.37879482 | 4.378795 | 69.459 | 16.26 | ** |
| |
5 | 0.31520518 | 0.063041 | |||
| Amount to | 6 | 4.694 |
The comparison of table 2 experiment degree of hydrolysis and theoretical degree of hydrolysis
| Sequence number measured value predicted value Y |
| 1 ?7.53 7.4794029 0.05059713 |
| 2 ?7.99 8.2604936 -0.2704936 |
| 3 ?8.66 8.7291481 -0.0691481 |
| 4 ?9.44 9.0415844 0.39841561 |
| 5 ?9.5 ?9.3540207 0.1459793 |
| 6 ?9.59 9.8226752 -0.2326752 |
| 7 ?9.8 ?9.8226752 -0.0226752 |
Claims (2)
1. new method of utilizing biology sensor fast measuring collagen hydrolysate degree; It is when utilizing the hydrolysis by novo macromolecular collagen protein to generate micromolecule polypeptide; Free aminoacid content increases gradually in the enzymolysis liquid; The amount of its increase and degree of hydrolysis size are proportionate, and therefore, calculate the size of degree of hydrolysis through total amino acid content or a certain amino acid content in the detection enzymolysis liquid; This method adopts the glucose oxidation enzyme biologic sensor, generates ketone acid and H according to glutamic acid and glucose oxidation enzyme reaction
2O
2Principle, the H of generation
2O
2With H in the biology sensor
2O
2The electrode contact produces current signal; This current signal and aminoglutaric acid concentration are linearly proportional; After MICROCOMPUTER PROCESSING, direct display result: concrete operations are to choose alkali protease, hydrolysis cod collagen under the condition of pH8.5,45 ℃ of hydrolysis temperatures, enzyme concentration 90mg/kg, concentration of substrate 2%; Get enzymolysis liquid 50 μ L; Inject glucose oxidation enzyme biologic sensor reaction tank, reading behind the 20s is according to the computing formula y=0.7811x+5.9172 calculating degree of hydrolysis of glutamic and degree of hydrolysis; Wherein x is a glutamic, and y is a degree of hydrolysis.
2. the new method of utilizing biology sensor fast measuring collagen hydrolysate degree according to claim 1; When adopting dglutamic oxidase biosensor assay glutamic; The glucose oxidation enzyme biologic sensor is a SBA-40C type bio-sensing analyser, and sample size is 50 μ L, the accounting equation y=0.7811x+5.9172 of glutamic and degree of hydrolysis; Wherein x is a glutamic, and y is a degree of hydrolysis; The dependence among equations coefficient | r|=0.9658413, the F value is 69.46.
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| CN2007100149618A CN101246140B (en) | 2007-06-16 | 2007-06-16 | Novel method for fast measuring collagen hydrolysis degree by biologic sensor |
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| CN2007100149618A CN101246140B (en) | 2007-06-16 | 2007-06-16 | Novel method for fast measuring collagen hydrolysis degree by biologic sensor |
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| CN101246140B true CN101246140B (en) | 2012-01-11 |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5093474A (en) * | 1988-08-04 | 1992-03-03 | Bar Ilan University | Process for the production of gelatin from fish skins |
| CN1328100A (en) * | 2001-06-26 | 2001-12-26 | 陈经国 | Hydrolytic collagen |
| CN1560270A (en) * | 2004-03-12 | 2005-01-05 | 中国海洋大学 | Process for preparing active peptide using fish skin collagen step directional enzynolysed by composit enzyme |
| CN1632528A (en) * | 2004-12-27 | 2005-06-29 | 山东大学 | Rapid determination technique for angiotensin converting enzyme inhibition activity of protein zymolyte |
| CN1749296A (en) * | 2005-10-11 | 2006-03-22 | 大连轻工业学院 | Acid soluble fish skin collagen and its preparing method |
| CN1749275A (en) * | 2004-09-15 | 2006-03-22 | 天津科技大学 | Method for processing hydrolytic fish skin collagen |
-
2007
- 2007-06-16 CN CN2007100149618A patent/CN101246140B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5093474A (en) * | 1988-08-04 | 1992-03-03 | Bar Ilan University | Process for the production of gelatin from fish skins |
| CN1328100A (en) * | 2001-06-26 | 2001-12-26 | 陈经国 | Hydrolytic collagen |
| CN1560270A (en) * | 2004-03-12 | 2005-01-05 | 中国海洋大学 | Process for preparing active peptide using fish skin collagen step directional enzynolysed by composit enzyme |
| CN1749275A (en) * | 2004-09-15 | 2006-03-22 | 天津科技大学 | Method for processing hydrolytic fish skin collagen |
| CN1632528A (en) * | 2004-12-27 | 2005-06-29 | 山东大学 | Rapid determination technique for angiotensin converting enzyme inhibition activity of protein zymolyte |
| CN1749296A (en) * | 2005-10-11 | 2006-03-22 | 大连轻工业学院 | Acid soluble fish skin collagen and its preparing method |
Non-Patent Citations (1)
| Title |
|---|
| 邓尚贵，杨萍.青鳞鱼肌肉蛋白复合酶水解机制研究.《食品科学》.2004,第25卷(第3期),67-75. * |
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