CN108181245A - The method and its kit of lactoperoxidase enzymatic activity in enzyme process quantitative determination breast - Google Patents
The method and its kit of lactoperoxidase enzymatic activity in enzyme process quantitative determination breast Download PDFInfo
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- G—PHYSICS
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Abstract
The present invention is improved the method for lactoperoxidase in enzymatic assays breast, it is prepared for stable standard solution, and improve the pre-treating method and post-processing approach of dairy food sample, it solves enzyme process quantitatively to detect in breast in lactoperoxidase enzymatic activity, standard curve is unstable, the technical issues of specificity and sensitivity of sample detection be not high, and the complete kit available for detection method is provided, facilitate the detection that Routine Test Lab carries out lactoperoxidase enzymatic activity in breast.
Description
Technical field
The invention belongs to enzymologys to analyze detection field, relates in particular to one kind and is quantitatively detected in breast for enzyme chemical method
Kit, preparation method and the corresponding assay method of lactoperoxidase enzymatic activity.
Background technology
Lactoperoxidase (Lactoperoxidase, EC 1.11.1.7, abbreviation LP) be peroxiredoxins into
One of member, is a kind of hemoprotein being present in milk, is made of skin chain more than one, including 612 amino acid residues, is divided
Son amount is about 78kD, and isoelectric point 9.6 is one of composition very common in human milk and cow's milk.
Lactoperoxidase and hydrogen peroxide and thiocyanate radical (SCN) can form " lacto-peroxidase system
(LPS)”.This enzyme system has bacteriostatic activity, can inhibit gram-positive bacteria and negative bacterium under conditions of no refrigeration
Growth, extend the shelf-life of fresh milk, have the function of " cold sterilization ".LPS in breast not only has antibacterial action, can also prevent
The accumulation of the peroxide such as hydrogen peroxide so as to avoid cellular damage caused by peroxide, is played the role of protecting mammary gland.
LP contents are especially abundant in colostrum.In addition, the destruction of LP is by one of index as pasteurization effect.
Therefore Dairy Industry needs to detect the activity of LP in breast, evaluation and fresh-keeping measure for LP activity in fresh milk
Processing；It is also used for the evaluation of dairy products sterility or understands the heat treatment situation of breast indirectly；Understood by measuring LP activity
Health status of milk cow etc. in certain period.
At present, the method both at home and abroad about lactoperoxidase detection in breast is few, mainly uses colorimetric method such as guaiaci lignum
Phenol method, ABTS methods or ELISA method etc., these methods are all there are various shortcomings, such as detection process complexity is cumbersome, detection knot
The problems such as fruit accuracy is poor.On the other hand, since the exact level of lactoperoxidase standard items is not easy to determine, and newborn peroxidating
Object enzyme stability is not high, the content of the standard solution of preparation be not easy to keep stablizing it is constant, also to lactoperoxidase in breast
Quantitative analysis brings uncertainty.
In addition, commercially available lactoperoxidase enzyme detection kit is substantially the detection reagent of ELISA method currently on the market
Box.On the one hand, lactoperoxidase therein is quantitatively detected using ELISA method, can not just be kept away containing miscellaneous enzyme in breast
Exempt from that there are the cross reactions between enzyme；On the other hand, protein of milk complicated component, lactoperoxidase is relative to other eggs
Content is extremely low for white matter, is measured using ELISA method very high to the sensitivity and specific requirements of method；And by being used
The antibody of lactoperoxidase active enzyme and the enzyme inactivated usually cannot be distinguished, lead to this method to the enzyme that has been passivated
Also there is a degree of reaction, so as to make the distortion of the result of ELISA method detection more serious.
Dairy industry still needs to exploitation and is suitable for method and its detection kit that lactoperoxidase quantitatively detects.
Invention content
For overcome the deficiencies in the prior art, the present inventor is from the stability of standard solution, sample treatment side
Method etc. is started with research, solves the technological deficiency that enzyme process quantitatively detects lactoperoxidase enzymatic activity in breast, so as to complete
The present invention.
The first aspect of the invention is to provide a kind of lactoperoxidase standard solution.The solution can be at 2~8 DEG C
Long-term preserve and lactoperoxidase therein is activity stabilized.The solution can be placed 30 days and newborn mistake therein at 20~37 DEG C
Peroxidase activity is stablized.Solve -20 DEG C of freezen protectives of lactoperoxidase standard solution needs in the prior art, repeatedly
Detection that freeze thawing is brought is inconvenient and frozen-thaw process in enzymatic activity the problem of reducing, also solve the newborn peroxidating of the prior art
Object enzyme standard solution is (generally 25~37 DEG C) rapid deactivation at a temperature of detection the problem of.
A kind of lactoperoxidase standard solution, contains lactoperoxidase, trehalose, soluble calcium salt and water.
It is preferred that the content ratio of wherein each component is：Lactoperoxidase：Trehalose：Soluble calcium salt in terms of calcium=1U：
(0.125g~0.25g)：(0.00025g~0.002g)；More preferably：Lactoperoxidase：Trehalose：Soluble calcium salt is with calcium
Meter=1U：(0.15g~0.25g)：(0.001g~0.0015g).
It is preferred that the content of lactoperoxidase is 10U/L~800U/L.Further preferably 320~800U/L.Most preferably
For 400U/L.
In one embodiment of the invention, the lactoperoxidase standard solution contains 400U/L breast peroxidating
Object enzyme, the content of trehalose is 5-10w/w%, and soluble calcium salt is using the content that calcium is counted as 0.01-0.08w/w%.It is preferred that seaweed
The content of sugar is 6-10w/w%, and soluble calcium salt is using the content that calcium is counted as 0.04-0.06w/w%.
According to the present invention, the soluble calcium salt can be selected from calcium chloride, calcium nitrate, calcium bicarbonate, calcium bisulfate, phosphoric acid
The common soluble calcium salt such as calcium dihydrogen, calcium monohydrogen phosphate it is one or more.
According to the present invention, the lactoperoxidase standard solution can be provided directly as the product in kit,
It is placed at 2~8 DEG C and preserves.
In an embodiment of the invention, using the lactoperoxidase standard solution of various concentration gradient as inspection
Reagent in test agent box, when detection directly use with establish in standard curve, such as kit pack upper 25,50,100,
200th, the standard solution of five concentration gradients of 400U/L, directly using establishing standard curve during detection.
In yet another embodiment of the present invention, by the lactoperoxidase standard solution of high concentration, (such as enzyme contains
Measure the solution for 320~800U/L) as the reagent in detection kit, before detection, which is diluted
The standard items for obtaining various concentration gradient are used for the foundation of standard curve.For example, it is tried 400U/L standard solutions as detection
Reagent in agent box provides, be diluted to 25 when detecting, 50,100,200,400U/L concentration gradients, for establishing standard
Curve.
The second aspect of the invention is to provide a kind of kit.The kit quantitatively detects newborn mistake in breast for enzyme process
Peroxidase activity,, can by color reaction by the method for absorbance measurement enzymatic activity suitable for utilizing enzymatic reaction principle
With ABTS (2 ' 2- azine groups-bis- (3- ethyl benzo thiophene pyrrolin -6- sulfonic acid) two ammonia salts), (3,3 ', 5,5 '-tetramethyl joins TMB
Aniline), guaiacol, aniline and o-phenylenediamine etc. are substrate, particularly preferably using TMB as substrate.
A kind of kit that lactoperoxidase enzymatic activity in breast is quantitatively detected for enzyme process includes sample treatment liquid, newborn mistake
Oxide enzyme standard solution and optional sample diluting liquid；The sample treatment liquid is used for the pre-treatment of dairy food sample, contains
There are water and the acetic acid of 10-20w/w%；The sample diluting liquid contains water and 1-5w/ for diluting treated dairy food sample
The BSA of w%.
According to the present invention, the lactoperoxidase standard solution contains lactoperoxidase, trehalose, solubility calcium
Salt and water.
It is preferred that the content ratio of each component is：Lactoperoxidase：Trehalose：Soluble calcium salt in terms of calcium=1U：
(0.125g~0.25g)：(0.00025g~0.002g)；More preferably：Lactoperoxidase：Trehalose：Soluble calcium salt is with calcium
Meter=1U：(0.15g~0.25g)：(0.001g~0.0015g).
Further preferably, the lactoperoxidase standard solution is a lactoperoxidase standard solution,
The content of middle lactoperoxidase is 320~800U/L, most preferably 400U/L.In an embodiment of the invention, institute
State lactoperoxidase standard solution in kit be containing 400U/L lactoperoxidases, 5-10w/w% trehaloses,
The lactoperoxidase standard solution of soluble calcium salts of the 0.01-0.08w/w% in terms of calcium；It is preferred that the content of trehalose is 6-
10w/w%, soluble calcium salt is using the content that calcium is counted as 0.04-0.06w/w%.
Further preferably, the lactoperoxidase standard solution be multiple, for example, at least 4, preferably at least 5,
Such as 5,6,7 lactoperoxidase standard solutions, the newborn mistake in the multiple lactoperoxidase standard solution
Oxide enzyme concentration is incremented by multiple.It is further preferred that the concentration incremental in multiple is using 10-25U/L as starting point；It is further preferred that institute
It states and is incremented by the incremental concentration of multiple at least 2 times of multiple.It is further preferred that the concentration gradient is with 25U/L lactoperoxidases
Content is starting point, and the multiple with 2 times is incremented by.In an embodiment of the invention, the lactoperoxidase in the kit
Enzyme standard solution is the standard solution of five concentration gradients of 25U/L, 50U/L, 100U/L, 200U/L, 400U/L.
It is preferred that acetic acid content is 10-15w/w% in the sample treatment liquid.
It is preferred that BSA contents are 1-3w/w% in the sample diluting liquid.
It is preferred that the kit further includes substrate solution, the substrate solution contain 0.1mol/L citric acids,
0.2mol/L disodium hydrogen phosphates, 0.3mmol/L H2O2With 0.3mmol/L TMB.
It is preferred that the kit further includes terminate liquid, the terminate liquid is the sulfuric acid solution of 0.18mol/L.In due course
It adds in terminate liquid and terminates enzyme reaction, and the TMB blue compounds generated through enzyme reaction can be changed into suitable for general microplate reader
The yellow compound measured under 450nm, the detection method and its kit for making the present invention can be suitably used for a variety of detecting instruments, can be into
One step improves the sensitivity measured and result accuracy.
It is preferred that the kit further include kit application method illustrate material.
According to the present invention, the above-mentioned various reagents in the kit are independently packaged in reagent bottle.In reagent bottle
The volume of each reagent can change in very large range, according to the quantity of the sample-loading amount of detection instrument and every batch of detection sample
It is adjusted.
According to the present invention, the configuration sample dilution such as deionized water, distilled water, pH common buffer solution close with breast can be used
Liquid, as long as the dicyandiamide solution can maintain the activity and function of BSA.The common buffer solution includes but not limited to, carbonic acid
Salt-bicarbonate buffer, PBS liquid etc..
For example, absorbance detection is carried out using microplate reader, the microwell plate that can be used microplate reader mating, such as 24 orifice plates, 48
Orifice plate or 96 orifice plates etc., detection sample-loading amount is small, then the volume of each reagent can be smaller in reagent bottle, such as in 1~60ml.Using
Spectrophotometer carry out absorbance detection, the volume of cuvette is larger, detection sample-loading amount it is big, then in reagent bottle each reagent volume
Can be larger, such as in 10~1000ml.
In an embodiment of the invention, it is included in the kit for microplate reader detection, 10 parts can be handled
The amount of reagent of every part of 10ml dairy food sample：LP standard solutions (the sea of the LP containing 400U/L, 6-10w/w% of 1 bottle of 1ml dress
Algae sugar, soluble calcium salts of the 0.04-0.06w/w% in terms of calcium), at the sample containing 10-15w/w% acetic acid of 1 bottle of 60ml dress
Manage liquid, the sample diluting liquid containing 1-3w/w%BSA of 1 bottle of 12ml dress, the substrate solution of 1 bottle of 6ml dress, the end of 1 bottle of 12ml dress
Only liquid.The kit can further contain one piece of 96 orifice plate.
To further improve reagent storage stability, LP standard solutions, substrate solution, sample diluting liquid preferably use brown
Or amber glass bottle contains；Preferably all of reagent is placed at 2~8 DEG C and preserves.
The third aspect of the invention is to provide a kind of method that enzyme process quantitatively detects lactoperoxidase enzymatic activity in breast.It should
Method carries out pre-treatment using sample treatment liquid to dairy food sample, reduces other albumen or enzyme and lactoperoxidase enzymatic is reacted
Interference, improve the specificity and sensitivity of detection；Further, this method establishes standard song using stable LP standard items
Line is conducive to improve the stability of testing result；Further, in order to improve the accuracy of detection, according to dairy products feature to sample
Carry out necessary dilution.
A kind of method that enzyme process quantitatively detects lactoperoxidase enzymatic activity in breast, includes the following steps：
1) sample preparation：Dairy food sample is taken, adds in sample treatment solution mixing, filtering supernatant after centrifugation collects filtrate,
Optionally filtrate is diluted with sample diluting liquid；
2) it detects：Substrate solution blending incubation is added in filtrate after each standard items and filtrate or dilution, adds in terminate liquid
Mixing reads OD values at 450 nm.
The sample treatment liquid contains water and the acetic acid of 10-20w/w%；It is preferred that acetic acid content in the sample treatment liquid
For 10-15w/w%.
The sample diluting liquid contains water and the BSA of 1-5w/w%；It is preferred that BSA contents are 1- in the sample diluting liquid
3w/w%.
The substrate solution contains 0.1mol/L citric acids, 0.2mol/L disodium hydrogen phosphates, 0.3mmol/L H2O2With
0.3mmol/L TMB。
The terminate liquid is the sulfuric acid solution of 0.18mol/L.
It is preferred that the method further includes results to calculate step, using the difference of standard items and the absorbance value of blank as Y
Axis, a concentration of X-axis structure standard curve of standard items, according to sample and the difference of the absorbance value of blank, reads from standard curve
The concentration (U/L) of LP in sample is taken, is then multiplied by extension rate.
If using the lactoperoxidase standard solution (such as 320-800U/L) of high concentration, the method is also
Further comprise the preparation process of standard items, the standard solution of high concentration is diluted to various concentration gradient with sample diluting liquid
Standard items, and using sample diluting liquid as blank control；The lactoperoxidase standard solution contains lactoperoxidase,
Trehalose, soluble calcium salt and water, the content ratio of each component are：Lactoperoxidase：Trehalose：Soluble calcium salt is in terms of calcium
=1U：(0.125g~0.25g)：(0.00025g~0.002g)；It is preferred that lactoperoxidase：Trehalose：Soluble calcium salt with
Calcium meter=1U：(0.15g~0.25g)：(0.001g~0.0015g).For example, 400U/L lactoperoxidases, 6- will be contained
The trehalose of 10w/w%, the lactoperoxidase standard solution of soluble calcium salts of the 0.04-0.06w/w% in terms of calcium, uses sample
Product diluted is 25,50,100,200,400U/L concentration gradients, for establishing standard curve.
When diluting filtrate with sample diluting liquid, extension rate is selected according to the property of dairy food sample, such as：Fresh milk or lactogenesis
Middle LP activity is higher, and filtrate carries out subsequent detection after can diluting 10-80 times；LP activity is weakened in pasteurization breast, filtrate
Subsequent detection is carried out after 5-30 times can be diluted；It is relatively low that ultra-high-temperature sterilized milk or super bar kill LP activity in breast, and filtrate can be without
Dilution is directly detected.
It is preferred that add in 0.3-0.8ml sample treatment liquids in per 10ml dairy food samples.
It is preferred that it is placed 5-10 minutes after adding in sample treatment liquid.
It is preferred that isometric substrate solution is added in filtrate after standard items, filtrate or dilution.
It is preferred that it is incubated 8-15 minutes at 20-25 DEG C after adding in substrate solution.
Detection is completed in 30 minutes after terminate liquid it is preferred that adding in.
It is preferred that it is detected using microplate reader.
The method of the present invention is limited to 6U/L for the detection of LP activity in dairy products stoste.
Detect and react according to enzyme process, the linear detection range of the kit described in the second aspect of the present invention for 0.0~
400U/L LP。
In the specific embodiment of the present invention, described method includes following steps：
1) sample preparation：
(1) it pipettes in 10ml milk samples to centrifuge tube, restores to room temperature.
(2) 0.6ml sample treatment solution, while slowly vibrating mixing are slowly added to, is placed at room temperature for 5-10 minutes.
(3) 3000g is centrifuged 10 minutes, with filter paper filtering supernatant.It is to be measured to collect filtrate.
(4) filtrate is diluted with sample diluting liquid.
2) prepared by standard items：400U/L standard solutions are subjected to gradient dilution with sample diluting liquid, prepare 200U/L,
The standard solution of 100U/L, 50U/L, 25U/L, and using sample diluent solution as blank (0U/L).
3) detecting step：
It takes out required amount of micropore to be placed on micropore frame, each standard items or sample to be tested correspond to a micropore, record
Standard items and sample position, all samples and standard items do 3 groups of Duplicate Samples.
Added in into micropore 50 μ L LP standard items (0,25,50,100,200,400U/L) or sample, often plus a standard
Product or sample replace a new suction nozzle.
50 μ l substrate solutions are added in into each micropore, are mixed, are incubated 10 minutes at 20-25 DEG C.
100 μ L terminate liquids are added in into each micropore, gently vibrates and is mixed in horizontal plane.Add in 30 minutes after terminate liquid
It is interior, read and record the OD values of each micropore at 450 nm using microplate reader.
4) result calculates
Calculate the mean absorbance values of each standard items and sample.The mean absorbance values of standard items and sample are subtracted into sky
The absorbance value of (0U/L) in vain.
The deviation of Duplicate Samples absorbance value is qualified data within 10%.
Using the difference of standard items and the absorbance value of blank as Y-axis, a concentration of X-axis structure standard curve of standard items.According to
The difference of the absorbance value of sample and blank reads the concentration (U/L) of LP in sample from standard curve, is then multiplied by dilution times
Number.
The mode of the present invention is suitable for the quantitative detection of the lactoperoxidase enzymatic activity of dairy food sample.The dairy products can be
Fresh milk, reconstituted milk, pasteurization breast, ultra-high-temperature sterilized milk, super bar is killed breast etc., can be cow's milk, and sheep is newborn, horse breast etc..
Beneficial effects of the present invention：
For the detection method and its kit of the present invention based on enzymatic reaction, enzymatic reaction specificity is stronger, reacts bottom
Object only can just make its discoloration by LP effects；It is active LP to measure, and metachromasia will not occur for the LP for being passivated inactivation；It is logical
Can directly the LP in sample be quantitative determined by crossing stable LP standard items；It provides containing sample treatment liquid, sample dilution
Liquid, stabilization standard solution kit, so as to simplify the disadvantages such as error caused by mensuration program and reagent preparation, carry
The high cheap property and stability of detection method.
In addition, OD can be read simultaneously preferably using microplate reader in the assay, avoid with during spectrophotometric determination because
The problem of OD values are inconsistent caused by time difference, and multiple samples are once can detect, working efficiency is greatly improved, is applicable in
It is used in common laboratory.
Description of the drawings
The examination criteria curve that Fig. 1 assay methods using the present invention are established with LP standard solutions
The 4 DEG C of decentralizations of different dilutions of Fig. 2 standard solutions 2 set to 0 the 450nm absorbance value schematic diagrames of -22 days, and abscissa is
Enzyme concentration, unit U/L.
The 37 DEG C of decentralizations of different dilutions of Fig. 3 standard solutions 2 set to 0 the 450nm absorbance value schematic diagrames of -22 days, abscissa
For enzyme concentration, unit U/L.
The 450nm absorbance value schematic diagrames of 7 days are placed at different 4 DEG C of the dilutions of Fig. 4 standard solutions 1 and 2, wherein top
Curve for standard solution 2, lower curve is standard solution 1, and abscissa is enzyme concentration, unit U/L.
Specific embodiment
The present invention is described further with reference to embodiments.It should be noted that embodiment cannot function as to this hair
The limitation of bright protection domain, it will be understood by those skilled in the art that, any improvements introduced on the basis of the present invention and variation all exist
Within protection scope of the present invention.
Chemical reagent used in following embodiment is all conventional reagent, commercially available.
It is prepared by reagent：
1) preparation of LP standard solutions：The LP standard items that contain exact level by determination of activity will be weighed and be put in sterilizing
50mL volumetric flasks in, add in the trehalose and CaCl of weighing2Afterwards, pH is adjusted to be 6.2 and be settled to scale, is configured to containing LP
400U/L, trehalose 6w/w%, calcium chloride 0.05w/w% in terms of calcium standard solution.It draws 1mL and is sub-packed in 2mL browns
In reagent bottle, it is put in 4 DEG C of preservations.
Before detection, standard solution is diluted, to obtain 25,50,100,200,400U/L concentration gradients, for building
Day-mark directrix curve.
2) preparation of LP substrate solutions：Citric Acid Mono (C is weighed respectively6H8O7·H2O) 21.014g, disodium hydrogen phosphate
(Na2HPO4) 28.392g, 3,3`, 5,5`- tetramethyl benzidines (TMB) 0.0721g be put into the beaker of 1L add in 900mL go from
Sub- water adjusts pH5.4 after dissolving.The solution is transferred in 1L volumetric flasks, suitable amount is added according to the content of hydrogenperoxide steam generator
Hydrogen peroxide, be settled to scale, be configured to containing：Citric acid (0.1mol/L)-disodium hydrogen phosphate (0.2mol/L)-H2O2
The LP substrate solutions of (0.3mmol/L)-TMB (0.3mmol/L), 4 DEG C of preservations.
3) sample treatment solution：The solution containing 10w/w% acetic acid with acetic acid and deionized water configuration.
4) sample diluent solution：The solution containing 2w/w%BSA with BSA and deionized water configuration.
5) terminate liquid：The sulfuric acid solution of 0.18mol/L.
Mentioned reagent is used in following embodiment.
The measure of lactoperoxidase in 1 lactogenesis of embodiment
1) sample treatment：
It pipettes in 10ml milk samples to centrifuge tube, restores to room temperature.0.6ml sample treatments are slowly added into centrifuge tube
Solution, while slowly vibrating.It is placed at room temperature for 5-10 minutes.It is centrifuged 10 minutes in 3000g, with filter paper filtering supernatant.Collect filter
Liquid is to be measured, with drawing appropriate filtrate after 0.45 μm of membrane filtration in 10mL finger-type pipes, with sample diluting liquid by 20 times, 40
Times, 80 times be diluted after measure.
2) drafting of standard curve
96 hole microwell plates are taken, 50 μ l LP standard solutions are separately added into micropore, concentration is respectively 0,25,50,100,
200,400U/L) 50 μ l substrate solutions, are added in into each micropore, are mixed, (20-25 DEG C) of room temperature is incubated 10 minutes.Again to every
100 μ l terminate liquids are added in a micropore, gently vibrate mixing in horizontal plane.It adds in 30 minutes after terminate liquid, uses microplate reader
The OD values of each micropore are read and recorded at 450 nm.Using OD values as ordinate, it is bent to draw standard using normal concentration as abscissa
Line calculates regression equation.
1 standard items measurement result of table
| Standard LP contents (U/L) | 0 | 25 | 50 | 100 | 200 | 400 |
| Light absorption value | 0.06 | 0.268 | 0.385 | 0.600 | 0.889 | 1.596 |
3) sample measures
The 50 μ l of sample liquid after dilution are drawn in micropore, by the operation identical with 2) step, add in substrate solution and termination
Liquid reads OD values.It is checked in by standard curve or the content (U/L) of LP in sample liquid is obtained by regression equation calculation.To same all one's life
3 dilutions of milk sample product carry out 6 subsynchronous experiments, as a result such as table 2.
The measurement result of lactoperoxidase in 2 same lactogenesis of table
The dilution being calculated by 2 result of table is respectively the relative standard of LP activity in 20,40,80 times of lactogenesis sample
Deviation RSD is 0.88%, 4.34%, 3.47%, and 6 times for illustrating while measuring meet methodology requirement without significant difference.
Although because of extension rate difference, there is some difference between different dilutions, and relative standard deviation RSD is
9.43%, 10% is less than, illustrates that the difference between different dilutions is not also notable, meets the requirement of methodology.
The light absorption value measured to different dilutions is analyzed：During 20 times of dilutions, light absorption value average out to 1.574 is fallen
The top of standard curve (light absorption value of curve peak (400U/L) is 1.596), this is certain to can quantitatively generate
Deviation；During 80 times of dilutions, light absorption value average out to 0.496 substantially at the middle part of standard curve, belongs to and model is most preferably quantified in detection
Most preferably to sample using 80 times of dilutions when enclosing, therefore measuring lactoperoxidase in lactogenesis.
Embodiment 2：Lactoperoxidase assay in pasteurization milk
By being carried out in embodiment 1, the dilution after sample treatment is measured continuous mode using 5 times, 10 times, 20 times.
6 subsynchronous experiments are carried out to same pasteurization milk sample, as a result such as table 3.
The measure of lactoperoxidase content in 3 same pasteurization milk of table
As seen from the results in Table 3, for pasteurization milk, the light absorption value of 5 times of dilutions has exceeded standard curve range, surveys
It is invalid to determine result.
The relative standard deviation RSD of 10 times and 20 times two dilutions is 0.71%, 1.53%, illustrates that same dilution is same
When measure 6 times between without significant difference, meet methodology requirement.Also, the RSD between 10 times and 20 times of extension rate is
3.40%, no significant difference also complies with methodology requirement.
The light absorption value measured to different dilutions is analyzed：During 10 times of dilutions, light absorption value average out to 1.404,
Be near the mark the peak (400U/L) of curve, can generate certain deviation to quantitative.During 20 times of dilutions, light absorption value average out to
0.824, substantially at the middle part of standard curve, belong to best quantification range in detection, therefore measure newborn peroxide in pasteurization milk
Most preferably to sample using 20 times of dilutions during compound enzyme.
Embodiment 3：Lactoperoxidase assay in ultra-high-temperature sterilized milk
Continuous mode does not do to dilute according to the characteristics of ultra-high-temperature sterilized milk, after sample treatment carrying out by being carried out in embodiment 1
It measures.
6 subsynchronous experiments are carried out to same ultra-high-temperature sterilized milk sample, as a result such as table 4.
The measure of lactoperoxidase content in 4 same ultra-high-temperature sterilized milk of table
| Serial number | 1 | 2 | 3 | 4 | 5 | 6 | Average value | RSD% |
| Light absorption value | 0.24 | 0.272 | 0.238 | 0.267 | 0.249 | 0.256 | 0.254 | - |
| LP(U/L) | 13.93 | 15.79 | 13.81 | 15.5 | 14.45 | 14.86 | 14.72 | 5.5161 |
As seen from the results in Table 4, for ultra-high-temperature sterilized milk, since lactoperoxidase has inactivated substantially, nothing in measure
Sample liquid need to be diluted again, the relative standard deviation RSD of 6 measurement results is 5.52%, and no significant difference, meeting methodology will
It asks.
The stability of 1 standard solution of comparative example
Following standard solution will be configured to deionized water by the LP standard items that determination of activity contains exact level：
Standard solution 1：LP 400U/L, pH6.2.
Standard solution 2：LP 400U/L, trehalose 6w/w%, the calcium chloride 0.05w/w% in terms of calcium, pH6.2.
Above-mentioned two standard solution is carried out to the dilution of various concentration, respectively obtains 25,50,100,200,400U/L is dense
After spending gradient, set to 0-22 days in 4 DEG C and 37 DEG C decentralizations respectively, each mark is during which measured using the method in 1 step 2) of embodiment
The absorbance value of quasi- product solution, with the stability of examination criteria product solution.Each concentration surveys 6 parallel samples.Statistical result table
Bright, each concentration of standard solution 2, which transfers the enzyme activity sex differernce for setting to 0 and being measured for -22 days at 4 DEG C and 37 DEG C, does not have conspicuousness,
Illustrate that the activity of wherein enzyme keeps stablizing, and (37 DEG C) are also very stable (referring to Fig. 2-3) under high temperature；And standard solution 1
There is the reduction with significant difference in the enzymatic activity that each concentration is measured after being placed 7 days at 4 DEG C, with standard solution 2
It compares, stability is bad (referring to Fig. 4).
The untreated liquid of 2 sample of comparative example is measured
Lactogenesis sample is taken, is pipetted in 10ml milk samples to centrifuge tube, is restored to room temperature.It is slowly added into centrifuge tube
0.6ml deionized waters, while slowly vibrating.It is placed at room temperature for 5-10 minutes.It is centrifuged 10 minutes in 3000g, supernatant is filtered with filter paper
Liquid.It is to be measured to collect filtrate, with appropriate filtrate is drawn after 0.45 μm of membrane filtration in 10mL finger-type pipes, is pressed with sample diluting liquid
20 times, 40 times, 80 times be diluted after measure.Thereafter it is operated according to the method and step of embodiment 1, as a result, it has been found that milk sample
Product colour developing unobvious, can not meet the requirement of detection.
Treated that sample is measured using deionized water dilution for comparative example 3
Lactogenesis sample is taken, is operated according to the method and step of embodiment 1, the difference lies in will be located using deionized water
Sample filtrate after reason is diluted by 20 times, 40 times, 80 times.As a result, it has been found that background is too high when microplate reader detects, can not also expire
The requirement detected enough.
Claims (9)
1. a kind of lactoperoxidase standard solution, contains lactoperoxidase, trehalose, soluble calcium salt and water；It is excellent
Choosing wherein each component content ratio be：Lactoperoxidase：Trehalose：Soluble calcium salt in terms of calcium=1U：(0.125g~
0.25g)：(0.00025g~0.002g)；Further preferred lactoperoxidase：Trehalose：Soluble calcium salt in terms of calcium=1U：
(0.15g~0.25g)：(0.001g~0.0015g).
2. lactoperoxidase standard solution as described in claim 1, the wherein content of lactoperoxidase for 10U/L~
800U/L；Further preferably 320~800U/L；Most preferably 400U/L.
3. lactoperoxidase standard solution as claimed in claim 2, the wherein content of lactoperoxidase are 400U/L,
The content of trehalose is 5~10w/w%；Soluble calcium salt counts content as 0.01-0.08w/w% using calcium；
It is preferred that the content of trehalose is 6-10w/w%；
It is preferred that soluble calcium salt counts content as 0.04-0.06w/w% using calcium.
4. a kind of kit that lactoperoxidase enzymatic activity in breast is quantitatively detected for enzyme process includes sample treatment liquid, newborn peroxide
Compound enzyme standard solution and optional sample diluting liquid；The sample treatment liquid contains water and the acetic acid of 10-20w/w%；Institute
It states sample diluting liquid and contains water and the BSA of 1-5w/w%；The lactoperoxidase standard solution contains lactoperoxidase,
Trehalose, soluble calcium salt and water, the content ratio of each component are：Lactoperoxidase：Trehalose：Soluble calcium salt is in terms of calcium
=1U：(0.125g~0.25g)：(0.00025g~0.002g), more preferably：Lactoperoxidase：Trehalose：Soluble calcium salt
In terms of calcium=1U：(0.15g~0.25g)：(0.001g~0.0015g)；
It is preferred that the lactoperoxidase standard solution is a lactoperoxidase standard solution, wherein newborn peroxidating
The content of object enzyme is 320~800U/L, most preferably 400U/L；Lactoperoxidase standard solution described in preferred reagent box
To contain 400U/L lactoperoxidases, 5-10w/w% trehaloses, the breast of soluble calcium salts of the 0.01-0.08w/w% in terms of calcium
Peroxidase standard solution, the content of more preferable trehalose is 6-10w/w%, and content of the soluble calcium salt in terms of calcium is
0.04-0.06w/w%；
It is preferred that the lactoperoxidase standard solution is at least five lactoperoxidase standard solution, described at least 5
Lactoperoxidase enzyme concentration in a lactoperoxidase standard solution is incremented by multiple；It is it is preferred that described in incremental dense of multiple
Using 10-25U/L as starting point, the multiple at least 2 times is incremented by degree；It is further preferred that it is described in the incremental gradient of multiple using 25U/L for
Point, the multiple with 2 times are incremented by；It is preferred that lactoperoxidase standard solution described in kit is 25U/L, 50U/L, 100U/
L, the standard solution of five concentration gradients of 200U/L, 400U/L；
It is preferred that acetic acid content is 10-15w/w% in the sample treatment liquid；
It is preferred that BSA contents are 1-3w/w% in the sample diluting liquid.
5. kit as claimed in claim 4, further includes substrate solution, the substrate solution contains 0.1mol/L lemons
Acid, 0.2mol/L disodium hydrogen phosphates, 0.3mmol/L H2O2With 0.3mmol/L TMB.
6. such as claim 4-5 any one of them kits, terminate liquid is further included, the terminate liquid is 0.18mol/L
Sulfuric acid solution.
7. such as claim 4-6 any one of them kits, it includes：The LP standard solutions of 1 bottle of 1ml dress, 1 bottle of 60ml dress
Acetic acid content be 10-15w/w% sample treatment liquid, the BSA contents of 1 bottle of 12ml dress are the sample diluting liquid of 1-3w/w%, 1
The substrate solution of bottle 6ml dresses, the terminate liquid of 1 bottle of 12ml dress, the LP standard solutions contain the LP of 400U/L, 6-10w/w%
Trehalose, soluble calcium salts of the 0.04-0.06w/w% in terms of calcium；It is preferred that the kit further contains one piece of 96 orifice plate.
8. a kind of method that enzyme process quantitatively detects lactoperoxidase enzymatic activity in breast, includes the following steps：
1) sample preparation：Dairy food sample is taken, adds in sample treatment solution mixing, filtering supernatant after centrifugation collects filtrate, optionally
Filtrate is diluted with sample diluting liquid；The sample treatment liquid contains water and the acetic acid of 10-20w/w%, and the sample diluting liquid contains
There are water and the BSA of 1-5w/w%；
2) it detects：Substrate solution blending incubation is added in filtrate after each standard items and filtrate or dilution, adds in terminate liquid mixing,
OD values are read at 450 nm；The substrate solution contains 0.1mol/L citric acids, 0.2mol/L disodium hydrogen phosphates, 0.3mmol/L
H2O2With 0.3mmol/L TMB；The terminate liquid is the sulfuric acid solution of 0.18mol/L；
It is preferred that the method further includes results to calculate step, and using the difference of standard items and the absorbance value of blank as Y-axis, mark
A concentration of X-axis structure standard curve of quasi- product, according to sample and the difference of the absorbance value of blank, reads sample from standard curve
The concentration (U/L) of middle LP, is then multiplied by extension rate；
It is preferred that if using the lactoperoxidase standard solution containing 320-800U/L LP, the method is also into one
Step includes the preparation process of standard items, with sample diluting liquid that the lactoperoxidase standard items containing 320-800U/L LP are molten
Liquid is diluted to the standard items of various concentration gradient, and using sample diluting liquid as blank control；The lactoperoxidase standard items
Solution contains lactoperoxidase, trehalose, soluble calcium salt and water, and the content ratio of each component is：Lactoperoxidase：Sea
Algae sugar：Soluble calcium salt in terms of calcium=1U：(0.125g~0.25g)：(0.00025g~0.002g)；
It is preferred that when diluting filtrate with sample diluting liquid, the filtrate of fresh milk or lactogenesis dilutes 10-80 times of rear progress subsequent detection；Bar
The filtrate of family name's sterile milk carries out subsequent detection after diluting 5-30 times；Ultra-high-temperature sterilized milk or the super bar of filtrate for killing breast are without dilution
Directly it is detected.
9. method as claimed in claim 8, wherein adding in 0.3-0.8ml sample treatment liquids in per 10ml dairy food samples；
It is preferred that it is placed 5-10 minutes after adding in sample treatment liquid；
It is preferred that isometric substrate solution is added in filtrate after standard items, filtrate or dilution；
It is preferred that it is incubated 8-15 minutes at 20-25 DEG C after adding in substrate solution；
Detection is completed in 30 minutes after terminate liquid it is preferred that adding in；
It is preferred that it is detected using microplate reader.
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Cited By (1)
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| CN110567947A (en) * | 2019-05-29 | 2019-12-13 | 北京泰德制药股份有限公司 | Kit for detecting activity of phosphatidized recombinant human superoxide dismutase |
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