CN110484598A - Application of the casein plate method in the quantitative analysis of proteinase activity - Google Patents
Application of the casein plate method in the quantitative analysis of proteinase activity Download PDFInfo
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- 230000000694 effects Effects 0.000 title claims abstract description 73
- 239000005018 casein Substances 0.000 title claims abstract description 59
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- 235000021240 caseins Nutrition 0.000 title claims abstract description 59
- 102000035195 Peptidases Human genes 0.000 title claims abstract description 29
- 108091005804 Peptidases Proteins 0.000 title claims abstract description 29
- 235000019833 protease Nutrition 0.000 title claims abstract description 13
- 238000004445 quantitative analysis Methods 0.000 title claims abstract description 8
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
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- 238000001514 detection method Methods 0.000 abstract description 24
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 5
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- 238000002835 absorbance Methods 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 108010079058 casein hydrolysate Proteins 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
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- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- NHJVRSWLHSJWIN-UHFFFAOYSA-N 2,4,6-trinitrobenzenesulfonic acid Chemical compound OS(=O)(=O)C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O NHJVRSWLHSJWIN-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
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- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
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- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
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- 238000000605 extraction Methods 0.000 description 1
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- 239000000706 filtrate Substances 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
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- 238000002372 labelling Methods 0.000 description 1
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- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-N o-dicarboxybenzene Natural products OC(=O)C1=CC=CC=C1C(O)=O XNGIFLGASWRNHJ-UHFFFAOYSA-N 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000004038 photonic crystal Substances 0.000 description 1
- -1 phthalic acid ester Chemical class 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
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- 238000002975 protease activity determination Methods 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229960004799 tryptophan Drugs 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/976—Trypsin; Chymotrypsin
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of application of casein plate method in the quantitative analysis of proteinase activity.The present invention utilizes prolease activity in casein plate method detection colored food, the result shows that casein plate method linear relationship is preferable, precision is preferable, repeatability is preferable, detection limit is lower, it is accurate to the testing result of colored samples prolease activity, a kind of new method is provided for prolease activity detection.The invention has the advantages that can effectively avoid due to the too deep caused error of color sample and measurement result inaccuracy, color sample, form are not required, it can be used for detecting light color or colourless Sample, it can also be used for detecting dark sample such as red heart dragon fruit etc., its is easy to operate, have a wide range of application, general testing requirements can be reached.The present invention can be effectively applied to the deeper product enzyme activity determination of the colors such as soy sauce, tealeaves.
Description
Technical field
The present invention relates to proteinase activity detection technique fields, the in particular to method of mixed fungus fermentation method preparation purple perilla jam.
Background technique
Proteolytic enzyme abbreviation protease is a kind of enzyme for carrying out protein hydrolysis, and being one group has height single-minded
The large complicated enzyme molecule of proteolysis function connects the peptide bond of amino acid that is, in the polypeptide chain by hydrolyzing to form protein,
Start the catabolism of protein.Assays for protease enzyme activity is research and a step important using protease.It is lured based on protease
The strategy of substrate cleavage is led, although colorimetric method and fluorescence method have obtained extensive development, light letter in protease activity determination
Number overlapping and complicated chemical labeling process limit the application of method.The detection of proteinase activity has using fluorescence method, but
There is the report based on the methods of Calorimetric Techniques, radioactivity, immunoassays, electrophoresis, ampere, optics.Ground substance assistant laser desorption/electricity
From flight time mass spectrum (MALDI-TOF-MS), probe or using silicon-based nanopore photonic crystal organic derivatization method come
Immobilization polypeptide, these detection methods require certain instrument and have certain operation requirement to experimenter, therefore are of little use.And
And many albumen enzymatic detection techniques need time-consuming progress sample preparation, this can have an adverse effect to the natural activity of enzyme.At present
Common proteinase activity detection method is colorimetric method.Before and after common proteinase activity mainly passes through detection enzyme hydrolysis, substrate
The reduction of protein concentration or the increase of product free amino acid or peptide concentration carry out quantitative expression.Such as ultraviolet specrophotometer
Method and biuret reagent are owned by France in the former, and ninhydrin, trinitrobenzene sulfonic acid method and forint phenol reagent process belong to the latter.
These method testing results are accurate, but testing result is also easy to produce error when detection colored food, is primarily due to ultraviolet spectrometry light
Degree meter is using light scattering principle, and when color sample is too deep, light absorption value is excessive, to generate error.Forint phenol method surveys protease
The principle of vigor are as follows: using casein as substrate, the amount for generating amino acid is measured afterwards for a period of time with example reaction, to calculate
Sample protein enzyme activity, the method is the disadvantage is that the factor for influencing reaction system is more, if containing citric acid, glycerol, sweet in sample
The objects such as propylhomoserin, Tris buffer, hyaluronic acid, dithiothreitol dithio, mercaptoethanol, ethylenediamine tetra-acetic acid (EDTA) and urea
Matter can generate interference effect；And it is influenced by protein specific and different proteins are because of tyrosine, tryptophane
Different and keep colored intensity slightly different, measurement result is easy to produce error.
Summary of the invention
For the deficiency of existing invention material, the present invention provides a kind of casein plate method quantifying in proteinase activity
Application in analysis, it is particularly suitable for detecting colored samples prolease activity, and precision is high, and repeatability is good, and has
There is the good rate of recovery.
In order to achieve the above object, the present invention is achieved by the following technical programs: casein plate method is in protease enzyme
Application in quantitative analysis living, the step of including the following steps: this method:
Step 1: casein plate is prepared: including casein 5g/L, glucose 10g/L, yeast extract 1g/L, phosphoric acid hydrogen two
Potassium 1g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.1g/L and agar 20g/L, in 121 DEG C of high pressure steam sterilization 20min；
Step 2: by culture medium down to culture dish, plate in homogeneous thickness is made；
Step 3: prepared casein plate is punched with 10mL pipette tips, three holes of a plate, as parallel,
It draws in 10 μ L enzyme solution injection holes, is put into 37 DEG C of culture carton upside down culture 1h-24h after standing 10min, take out its hydrolysis circle of measurement
Diameter simultaneously calculates dissolution circle area；Business trypsase is suitably diluted to the solution for being configured to required enzyme activity, respectively in junket egg
Punching culture is carried out on white plate, using dissolution circle area as abscissa (x, mm2), business trypsase vigor is ordinate (y, U
× mL-1) make business trypsase standard curve；
Step 4: sample enzyme activity is measured by standard curve step, and sample is hydrolyzed circle area and business tryptose
Enzyme standard curve is calculated, and corresponding sample to be tested enzyme activity is obtained.
Protease can hydrolyze the plate using casein as substrate, form observable hydrolysis circle.Casein hydrolysis circle holds
Easily observation, hydrolysis circle area and proteinase activity are positively correlated, can be substantially not by Color influences.Based on this, it is flat to construct casein
Plate method carries out quantitative analysis to prolease activity in colored food.
It being diluted with business trypsase, is configured to different vigor solution and is cultivated, measurement result makees standard curve,
Circle size is hydrolyzed according to sample, calculates sample vigor, inquires into and utilizes the feasible of casein plate quantitative analysis prolease activity
Property.The building of this method is particularly suitable for the vigor quantitative analysis of protease in colored food.
Compared with the existing technology, the present invention detects prolease activity in colored food using casein plate method to the present invention,
The result shows that casein plate method linear relationship is preferable, precision is preferable, repeatability is preferable, and detection limit is lower, to colored samples
The testing result of prolease activity is accurate, provides a kind of new method for prolease activity detection.The invention has the advantages that can be effective
Avoid due to color sample it is too deep caused by error and measurement result inaccuracy, color sample, form are not required, can
For detecting light color or colourless Sample, it can also be used to detect dark sample such as red heart dragon fruit etc., easy to operate, application range
Extensively, general testing requirements can be reached.The present invention can be effectively applied to the deeper product enzyme activity of the colors such as soy sauce, tealeaves and survey
It is fixed.
Detailed description of the invention
Fig. 1 is trypsase standard curve and tyrosine standard curve；
Fig. 2 is the detection of proteinase activity in colourless Sample；
In Fig. 2, a: casein plate is commercialized trypsin hydrolysis circle；B: casein plate sterile water hydrolysis；C: forint
Phenol method is compared with casein plate method；
Fig. 3 is colored samples enzyme activity determination；
In Fig. 3, a: inactivation soy sample is hydrolyzed in casein plate schemes；B: soy sample is hydrolyzed in casein plate schemes；
C: casein method and Forint phenol method detect colored food
Specific embodiment
1 Forint phenol method of embodiment and casein plate method compare
(1) casein plate method: prepared casein plate is punched with 10mL pipette tips (three holes of a plate,
As parallel), it draws in 10 μ L enzyme solution injection holes, is put into 37 DEG C of culture carton upside down culture 18h after standing 10min, takes out measurement
Its hydrolytic circle simultaneously calculates dissolution circle area.Business trypsase is suitably diluted to the solution for being configured to required enzyme activity, point
Punching culture is not carried out on casein plate, using dissolution circle area as abscissa (x, mm2), business trypsase vigor is vertical
Coordinate (y, UmL-1) makes business trypsase standard curve.Sample enzyme activity is measured by standard curve step, by sample
Product hydrolysis circle area is calculated with business trypsase standard curve, obtains corresponding sample to be tested enzyme activity.
(2) it Forint phenol method: is measured by GB/T23527-2009 " protease preparation ".
(3) two methods testing result compares
Fig. 1 (a) is the standard curve for surveying prolease activity, regression equation Y=0.016X+0.0098, R2=
0.9998, wherein Y is absorbance, and X is tyrosine concentration (μ g/mL).Using regression equation, calculate when absorbance is 1
Tyrosine concentration (μ g/mL), i.e. extinction constant K (should be in 95~100 ranges), this experiment K be 99.Fig. 1 (b) is tryptose
Enzyme standard curve, regression equation Y=0.5776X-9.0242, R2=0.9984, wherein Y is business trypsase vigor
(UmL-1), X is hydrolysis circle area (mm2).According to GB/T27404-2008 " Good Laboratory control specification ", method is confirmed
Regression coefficient requires 0.99, and this standard curvilinear regression coefficient is 0.9984, complies with standard, and show in a wider range, hydrolysis
There are preferable linear relationships between circle area and prolease activity.
Business trypsin solution caseinhydrolysate plate such as Fig. 2 (a, b) can obviously observe addition trypsin solution
There is round and smooth and contour edge and clearly hydrolyzes circle in the casein plate reacted, and sterile water caseinhydrolysate plate does not go out
Now hydrolysis circle, illustrates that it does not have prolease activity.The error generated when hydrolysis circle is round and smooth, edge obviously can effectively reduce measurement,
Obtain more accurate measurement result.
The commercial protease solution of 25U/mL is measured with Forint phenol method and casein plate method respectively, such as Fig. 2
(c).It can be seen that two methods testing result is closer to, Forint phenol method measurement result is 24.14U/mL, casein plate method
Measurement result is 24.01U/mL.Forint phenol method has centainly as the widest prolease activity detection method of current application
Representativeness, casein plate method for business trypsase testing result and Forint phenol method testing result it is about the same (p >
0.05), illustrate that casein plate method testing result is more accurate credible.
2 casein plate method precision of embodiment
18 parts of 0.100g business trypsase are accurately weighed, sterile water dissolution is added and is settled to 10mL, is configured to 250U/mL
Trypsin solution is diluted to 6 parts of 10UmL-1,25UmL-1,50UmL-1 solution respectively.10 μ L injection is drawn respectively
Casein plate is stood after 10min in 37 DEG C of culture carton upside down culture 18h, measures its hydrolytic circle, calculating enzyme activity and
RSD value.
1 low concentration of table, middle concentration and high concentration measurement result and RSD value
The results are shown in Table 1 for the precision of this method.Precision is 1.22%-8.07%, respectively less than 10%, under low concentration
Its RSD range is 2.92%-8.07%, less than 10%；Its RSD range is 0.45%-4.86% under middle concentration, less than 10%；
Its RSD range is 1.22%-2.85% under high concentration, less than 10%.Hao Wu is contained using HPLC-UV measurement phthalic acid ester
Amount, precision range are 3.9%~5.7%, and this method precision is 1.22%-8.07%, and more slightly higher compared with its, this is main
It is to have certain operating error because this experiment is manual measurement, and Hao Wu is measured using instrument, is wanted to precision
Ask higher.The above result shows that the method measurement result RSD value is respectively less than 10%, it may be said that bright this method precision is preferable.
3 casein plate method of embodiment detection limit
0.1g business trypsase is accurately weighed, 10mL water is dissolved in, is configured to 250U/mL solution.It is dense that 1mL or more is drawn again
Solution is spent, 25 times is diluted, is configured to 10U/mL solution.Be configured to respectively again 1U/mL, 2U/mL, 3U/mL, 4U/mL, 5U/mL,
6U/mL, 7U/mL, 8U/mL, 9U/mL, 10U/mL draw 10 μ L injection casein plate respectively, train after standing 10min in 37 DEG C
Carton upside down culture 18h is supported, its hydrolytic circle is measured, calculates enzyme activity and RSD value, in parallel three times.As a result such as 2 institute of table.
The detection limit experimental result of table 2
According to experimental result, can be detected when prolease activity is 7U/mL more accurate as a result, detected value is respectively
7.115,7.115,6.604U/mL, average value 6.945U/mL, standard deviation 0.295, relative standard deviation 4.25% are less than
10%, it is as a result more accurate.And when prolease activity is 6U/mL, measurement result is close to 6U/mL, but its RSD is
8.57%, error is larger, and measurement result is easy to produce error, so method detection is limited to 7U/mL.
4 casein plate method repeatability of embodiment
6 parts of 0.100g business trypsase are accurately weighed, sterile water dissolution is added and is settled to 10mL, is configured to 250U/mL
Trypsin solution.250U/mL trypsin solution 1mL is drawn, dilutes 10 times, is 25U/mL.10 μ L injection junket is drawn respectively
Albumen plate stands after 10min in 37 DEG C of culture carton upside down culture 18h, measures its hydrolytic circle, calculating enzyme activity and RSD
Value.Its enzyme activity is measured using casein plate method to 25U/mL protein enzyme solution, the results showed that measured value RSD is in 2.1%-
Between 5.73%, less than 10%, illustrate that result is more accurate.RSD is an important indicator for showing method accuracy, this method
Reproducible, error is small, and testing result is accurate and reliable.
3 repeated experiment result of table
The measurement of 4 colored food prolease activity of embodiment
Dry 1g mulberries, dragon fruit, violet cabbage and red three-coloured amaranth sample are weighed, 10mL PBS buffer solution is added and is extracted for 24 hours
(soy sauce is directly detected).10 μ l enzyme solutions are injected in casein plate punching, carry out inversion culture for 37 DEG C after placement 10min
18h.It measures its hydrolytic circle and calculates its enzyme activity and RSD value.
Sample qualitative filter paper at a slow speed after PBS extraction for 24 hours filters, and filtrate is centrifuged 10min at 8000r/min,
Make bacterial sediment, obtains crude enzyme liquid.Draw 10 μ L sample crude enzyme liquids injection punching casein plate, stand 10min after in 37
DEG C culture carton upside down culture 18h, take out measure its hydrolytic circle simultaneously calculate its area and enzyme activity.And good fortune is carried out to extracting solution
Woods phenol method measures prolease activity, as a result sees Fig. 3.
Soy sample caseinhydrolysate plate such as Fig. 3, obviously hydrolyzing, which does not occur, in Fig. 3 (a) to be enclosed, and illustrates that this sample does not have albumen
Enzyme activity fails substrate casein to be hydrolyzed effect, therefore does not generate hydrolysis circle；There is obvious hydrolysis circle in Fig. 3 (b), shows
This sample produces certain hydrolysis to substrate casein, hydrolyzes a certain range of casein around point of sample, therefore goes out
Now obvious hydrolysis circle.Hydrolysis circle area is calculated by measurement hydrolytic circle, can be counted further according to business trypsase standard curve
Calculate sample protein enzyme activity.
Forint phenol method is dry to colored samples such as mulberries and the measurement of the prolease activities such as red heart dragon fruit and casein plate
Method detection difference is larger, as a result such as Fig. 3 (c).When measuring tobacco leaf protein enzyme activity using Forint phenol method, the dry extracting solution sample of mulberries
Color is too deep, and color is black-and-blue after the reaction such as forint phenol reagent, measures its light absorption value through ultraviolet-uisible spectrophotometer
It is 4.000, has reached spectrophotometer maximum value, the reason of this is primarily due to stray light makes to analyze test result seriously inclined
From langbobier law, analytical error can be made to increase, cause result inaccurate.This is to the inspection of colored samples, that is, mulberries dry-eye disease
It surveys, 10 times of dilutions is carried out to extracting solution, obtaining solution colour is orange.Forint phenol method is carried out to this solution and surveys protease, is obtained
Reaction solution be it is black-and-blue, by measuring its light absorption value, discovery blank tube is higher than sample cell light absorption value, this is primarily due to stray light
The reason of, when generating sample solution concentration increase, the abnormal phenomenas such as absorbance value reduces instead.Stray light may make to analyze test result
Data it is less than normal, it is also possible to it is bigger than normal；Measurement data is less than normal if stray light other than test wavelength is absorbed by sample, this is this reality
Test the main reason for sample cell light absorption value is smaller than blank tube light absorption value.It is coloured that dragon fruit and soy sauce etc. are determined using Forint phenol method
The activity of protease in food.Dragon fruit, soy sauce, violet cabbage, three-coloured amaranth relative standard deviation be respectively 131.32%,
55.13%, 27.2% and 10.10%.Relative standard deviation shows to detect proteinase activity in colored food with Forint phenol method
Error is larger.However, this method do not detect mulberries it is dry in protease activity, illustrate this method the result is that mistake,
It is insecure.The proteinase activity of six kinds of colored food extracts is determined using casein plate method.As a result relative standard
For deviation between 4.265-10.35%, error is smaller, as a result accurately and reliably.
5 colored samples prolease activity mark-on reclaims of embodiment
Mark-on is carried out to soy sauce, red heart dragon fruit, violet cabbage, red three-coloured amaranth and mulberries dry-eye disease, 10U/mL business pancreas is added
Protein enzyme solution is drawn in 10 μ L mixing liquids injection casein plate hole, cultivates 18h in 37 DEG C of inversions.It takes out, it is straight to measure it
Diameter calculates hydrolysis circle area and enzyme activity.
Forint phenol method mark-on carries out recovery testu using 50 μ g/mL tyrosine solutions.Draw 0.1mL tyrosine solution
In test tube, it is measured according to Forint phenol method detection prolease activity.Recovery of standard addition=(mark-on style measured value-sample
Measured value)/scalar quantity * 100%.
Forint phenol method and casein plate method are shown in Table 4 to the rate of recovery of 5 kinds of colored foods.The casein plate method rate of recovery is
92.26%-97.84%, relative standard deviation 3.91%-10.88%.And the rate of recovery of Forint phenol method is 0.99%-
4.21%, relative standard deviation 17.59%.And the recovery of standard addition that Forint phenol method measures red dragon fruit is 4.21%, phase
It is 17.59% to standard deviation；The recovery of standard addition of violet cabbage be 0.99%, relative standard deviation 112.31%, this be because
It is greater than sample detection value for standard error.But enzymatic activity is not detected in mulberries dry-eye disease, is not also detected in soy sauce mark-on sample
To enzymatic activity, therefore the dry rate of recovery with soy sample of mulberries can not be calculated.When measuring red three-coloured amaranth recovery of standard addition, standard is found
The activity for adding enzyme is 0.446 ± 0.35U/ml, and the activity of sample is 1.386 ± 0.14U/ml, and mark-on sample enzyme activity is
0.446 ± 0.35U/ml, the activity of mark-on sample enzyme are less than the enzymatic activity of sample, can not calculate the rate of recovery.It is examined with Forint phenol method
The resultant error for surveying colored food is larger, thereby increases and it is possible to can not detect enzyme activity.
According to GB/T27404-2008 " Good Laboratory control specification-food physical and chemical analysis ", by thick constituent content <
When 0.1mg/kg, recovery of standard addition should between 60~120%, the casein plate method test sample rate of recovery be 97.8%~
107.49%, in this section, it is more accurate to illustrate that the method measures prolease activity；And Forint phenol method detects red heart flue
When fruit sample, the rate of recovery is only 4.21%, not up to requirements of the national standard, illustrates that Forint phenol method carries out colored samples protease
When vitality test, error is larger, as a result inaccurate.And when using Forint phenol method detection soy sample, although soy sample
10 times are diluted to orange from brownish black, but the sample cell light absorption value situation low compared with blank tube light absorption value, explanation occur
Color is more serious to Forint phenol method interference, and when detecting colored samples prolease activity using Forint phenol method, result is unreliable.
4 colored food sample mark-on reclaims of table
Known enzyme activity trypsin solution is detected with casein plate method by using Forint phenol method, the results showed that
Casein plate method testing result is accurate；To casein plate method detection precision, detection limit, repeated experiment, the results showed that
Precision, repeatability are good, and detection is limited to 7U/mL.By using Forint phenol method and casein plate method to actual sample such as mulberry
Shen dry, soy sauce, red heart dragon fruit etc. carry out the quantitative determination of protease activity survey, and detect its recovery of standard addition, the results showed that junket
It is more accurate that albumen flat band method safety check is surveyed, and error is smaller, and the rate of recovery is preferable.The rate of recovery of 6 kinds of colored samples is in 92.26%-
Between 107.49%, relative standard deviation is between 3.91%-10.88%.Casein plate method measurement result is with higher
Accuracy.
Illustrate the present invention can effectively avoid due to color sample it is too deep caused by error and measurement result inaccuracy, for
Color sample, form do not require, and can be used for detecting light color or colourless Sample, it can also be used to detect dark sample such as red heart flue
Fruit etc., it is easy to operate, have a wide range of application, general testing requirements can be reached.The present invention can be effectively applied to soy sauce, tealeaves etc.
The deeper product enzyme activity determination of color.
Embodiment of above is only specific example of the invention, it is clear that of the invention realizes not by the limit of aforesaid way
System.As long as using the inventive concept and technical scheme of the present invention carry out unsubstantiality improve, or it is not improved will be of the invention
Conception and technical scheme directly apply to other occasions, within the scope of the present invention.
Claims (1)
1. application of the casein plate method in the quantitative analysis of proteinase activity, characterized by the following steps: we
The step of method:
Step 1: casein plate is prepared: including casein 5g/L, glucose 10g/L, yeast extract 1g/L, dipotassium hydrogen phosphate 1g/
L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.1g/L and agar 20g/L, in 121 DEG C of high pressure steam sterilization 20min；
Step 2: by culture medium down to culture dish, plate in homogeneous thickness is made；
Step 3: prepared casein plate is punched with 10mL pipette tips, three holes of a plate, as parallel, absorption
In 10 μ L enzyme solution injection holes, 37 DEG C of culture carton upside down culture 1h-24h are put into after standing 10min, takes out and measures its hydrolytic circle
And calculate dissolution circle area；Business trypsase is suitably diluted to the solution for being configured to required enzyme activity, it is flat in casein respectively
Punching culture is carried out on plate, using dissolution circle area as abscissa (x, mm2), business trypsase vigor be ordinate (y, U ×
ML-1 business trypsase standard curve) is made；
Step 4: sample enzyme activity is measured by standard curve step, and sample is hydrolyzed circle area and business trypsase mark
Directrix curve is calculated, and corresponding sample to be tested enzyme activity is obtained.
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| CN114480564A (en) * | 2021-11-16 | 2022-05-13 | 鹤山市东古调味食品有限公司 | Method for measuring enzyme activity in soy sauce fermentation process |
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