CN103616454A - Method and kit for quantitatively detecting human beta-casein content - Google Patents
Method and kit for quantitatively detecting human beta-casein content Download PDFInfo
- Publication number
- CN103616454A CN103616454A CN201310656063.8A CN201310656063A CN103616454A CN 103616454 A CN103616454 A CN 103616454A CN 201310656063 A CN201310656063 A CN 201310656063A CN 103616454 A CN103616454 A CN 103616454A
- Authority
- CN
- China
- Prior art keywords
- casein
- beta
- special
- solution
- people
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention relates to the field of analytical chemistry, and discloses a method and a kit for quantitatively detecting human beta-casein content. According to the method and the kit, by utilizing a specific peptide sequence VMPVLK obtained after enzymolysis of human beta-casein, isotope labeled specific peptide and a sequence of an isotope labeling internal standard substance are designed, and a new accurate quantitative detection method is provided based on an internal standard method for quantitatively detecting the human beta-casein, with high accuracy, high precision and high sensitivity.
Description
Technical field
The present invention relates to analytical chemistry field, relate in particular a kind of method and kit of quantitative detection people beta-casein content.
Background technology
As far back as the eighties in last century, breastfeeding advantage is found by a lot of academic institutions in the world, and is had a lot of papers to confirm its superiority.Breast milk provides balanced protein to baby, fat, and carbohydrates, minerals and vitamins, can grow up healthy and sound baby.
Breast milk is the golden standard of formula milk.Due to the difference between milk and the nutritional labeling of breast milk, must in milk powder, add various nutriments, make it content and approach breast milk.The most important with protein in these nutriments, it plays the important and pivotal role in infant growth process.Breast milk proteins matter is comprised of 40% casein and 60% lactalbumin, and wherein casein is mainly comprised of beta-casein.From content, the beta-casein in breast milk and ALA are important nutrient proteins.So be necessary to set up one, quantitative detecting method is in order to detect the content of beta-casein in breast milk accurately, and its quantitative testing result can provide the theoretical foundation of protein reasonable constituents in formula milk.
The quantitative detecting method of protein mainly contains the methods such as liquid phase chromatography, capillary electrophoresis, gel molecular size exclusion chromatograph and euzymelinked immunosorbent assay (ELISA) both at home and abroad.First three class methods has all adopted UV-detector, but with the interference albumen that people's beta-casein absorption optical wavelength is close is a lot, causes its detection sensitivity low, selectivity is low, be not suitable for for quantitatively detecting the such matrix complexity of breast milk, the sample that kinds of protein is many.Enzyme linked immunological utilizes antigen and antibody specific binding ability to detect protein.Owing to lacking standard items, and the cross reaction of antibody to other breast milk proteins matter, the accurate quantitative analysis that makes euzymelinked immunosorbent assay (ELISA) also cannot be applied to breast milk proteins matter detects.
Summary of the invention
In view of this, the invention provides a kind of method and kit of quantitative detection people beta-casein content, make described method to detect beta-casein content in breast milk by accurate quantitative analysis, make sensitivity, precision and the matrix effect of described method meet existing standard.
For achieving the above object, the invention provides following technical scheme:
A method for quantitative detection people beta-casein content, comprises the steps:
Step 1, by 50mmol/L ammonium bicarbonate soln dilution for breast milk sample to be measured, get 10 μ L diluted breast milk samples, then add 20 μ mol/L Isotopic Internal Standard thing solution 10 μ L, 50mmol/L dithiothreitol (DTT) solution 10 μ L and 50mmol/L ammonium bicarbonate soln 945 μ L react, after reaction, add 150mmol/L iodo-acetamide solution 10 μ L dark places standing, then add 200 μ g/mL bovine trypsin solution 10 μ L enzymolysis, add after the pure formic acid of 5 μ L by high performance liquid chromatography triple level Four bar Mass Spectrometer Method of connecting, with internal standard method, obtain the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope,
Step 2, get each 10 μ L of the special poly saccharide peptide standard product solution of people's beta-casein of gradient concentration, after adding respectively the special poly saccharide peptide standard product solution 10 μ L of 20 μ mol/L isotope, with 50mmol/L ammonium bicarbonate soln 975 μ L, dilute, finally add the aqueous formic acid that 5 μ L percents by volume are 0.1%, by high performance liquid chromatography triple level Four bar Mass Spectrometer Method of connecting, the typical curve of the peak area ratio that obtains the special peptide of people's beta-casein and the special peptide of isotope and the corresponding special poly saccharide peptide standard product solution concentration of people's beta-casein, the special peptide of people's beta-casein that step 1 is obtained and the peak area ratio substitution typical curve of the special peptide of isotope obtain the concentration of the special peptide of beta-casein in breast milk sample, then be calculated as follows and obtain people's beta-casein content,
The special peptide volumetric molar concentration * people of beta-casein beta-casein molal weight * breast milk Sample Dilution multiple in people's beta-casein content=breast milk sample;
Wherein, the polypeptide that described Isotopic Internal Standard thing is sequence as shown in SEQ ID NO:1 and from N end the valine of the 9th and the 10th 's leucine by the full isotope labeling of carbon nitrogen, as follows:
YTKGRVMPV
*l
*kSPTIPFFDPQIPKL, wherein
*be expressed as the full isotope labeling of carbon nitrogen;
The special poly saccharide peptide standard product protein sequence of the special poly saccharide peptide standard product of described beta-casein and isotope is the polypeptide of sequence as shown in SEQ ID NO:2, and the special poly saccharide peptide standard product sequence of the isotope valine of the 4th and the 5th 's from N end leucine is by the full isotope labeling of carbon nitrogen.As follows:
VMPVLK；
VMPV
*l
*k, wherein
*be expressed as the full isotope labeling of carbon nitrogen.
The method of the invention is not limited to above-mentioned recorded numerical value, and those skilled in the art also can quantitatively detect and on principle basis, change other numerical value in the present invention.Breast milk Sample Dilution multiple in computing formula of the present invention comprises all dilution of breast milk sample to be measured operation, at the beginning with ammonium bicarbonate dilution and the follow-up dilution producing after mixing with all reaction solutions.
Wherein, the present invention is as preferably, 50mmol/L ammonium bicarbonate soln 1:9 dilution by volume for described breast milk sample to be measured.As example, the extension rate in computing formula is illustrated, be that breast milk sample to be measured dilutes 10 times at the beginning, and in follow-up detection, get the breast milk example reaction to be measured of 10 μ L dilutions, total reaction volume is 1000 μ L, 100 times have been diluted, total extension rate is 1000 times, and the extension rate in computing formula of the present invention should be 1000.
In addition, typical curve that the inventive method is done calculates for convenience of substitution, can carry out linear regression, draws linear equation Y=kX+b, and wherein Y is the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope; X is the concentration of the special peptide of people's beta-casein; K is the slope of linear equation; B is the intercept of linear equation.
Owing to worldwide lacking people's beta-casein standard items, the present invention is with bovine trypsin enzymolysis breast milk, in cutting peptide section, the numerous breast milk enzymes that produce select the peptide section that can only be produced by people's beta-casein enzymolysis, the present invention therefrom selects suitable peptide section, and according to the amino acid sequence of this peptide section, design the sequence of isotope-labeled special peptide and isotope labeling internal standard compound, and provide a kind of brand-new accurate quantitative analysis detection method based on internal standard method, solved the defect of existing detection method.
In quantitative detecting method of the present invention and kit, the special poly saccharide peptide standard product of beta-casein refers to the peptide section that people's beta-casein produces after enzymolysis through screening, i.e. the polypeptide of sequence shown in SEQ ID NO:2, VMPVLK; It is one of special peptide section of people's beta-casein, utilizes BLAST searching functions contrast internet database and use high resolution liquid chromatography tandem mass spectrometry to detect qualification result to show: be present in other protein in breast milk or other dairy products and do not have amino acid sequence and the trypsin digestion peptide section with this consensus amino acid sequence.This amino acid sequence is people's beta-casein peculiar peptide section after trypsin digestion.After chemosynthesis, purification, purity more than 99.0%, is used as Criterion curve in quantitative detecting method of the present invention;
The special poly saccharide peptide standard product of isotope-labeled people's beta-casein be one according to the special peptide sequence of people's beta-casein, after chemosynthesis with the amino acid whose special peptide section of isotope labeling, be called in the present invention the special peptide of isotope.Its amino acid sequence is VMPV
*l
*k, wherein V
*and L
*be respectively the complete isotope-labeled valine of carbon nitrogen and leucine, through synthetic, purify after purity more than 97.0%, and wherein there is not the special peptide of people's beta-casein.In quantitative detecting method of the present invention, as Criterion curve, use;
In isotope-labeled people's beta-casein, mark is special in quantitative measurement designs and synthetic internal standard compound, is called in the present invention Isotopic Internal Standard thing.The amino acid sequence of Isotopic Internal Standard thing as shown in SEQ ID NO:1 and valine and leucine by the full isotope labeling of carbon nitrogen, be specially YTKGRVMPV
*l
*kSPTIPFFDPQIPKL, wherein V
*and L
*for the complete isotope-labeled amino acid of carbon nitrogen.This peptide section has the enzymolysis efficiency same with people's beta-casein, can obtain the special peptide of isotope of equivalent, can do accurate quantitative analysis to the people's beta-casein in sample.Through synthetic, purify after purity more than 97.0%, and in mensuration process, do not produce special peptide.In quantitative detecting method of the present invention, as internal standard compound matter, use.
As preferably, described bovine trypsin solution is formulated by hydrochloric acid and bovine trypsin, described bovine trypsin specific activity >3000unit/mg protein.
As preferably, described enzymolysis is at 37 ℃ of constant temperature enzymolysis 2h.
As preferably, the special poly saccharide peptide standard product solution of people's beta-casein of described gradient concentration is prepared in accordance with the following methods:
Get respectively the special peptide solution 1 μ L of 1mmol/L beta-casein, 10 μ L, 20 μ L, 30 μ L, 40 μ L and 50 μ L, add ammonium bicarbonate soln to 1mL.
As preferably, reaction is at 60 ℃ of isothermal reaction 30min described in step 1.
As preferably, described high performance liquid chromatography separation condition:
Chromatographic column is C18 chromatographic column, and column temperature is 40 ℃; Mobile phase A is that percent by volume is the acetonitrile solution of 0.1% formic acid, and Mobile phase B is that percent by volume is 0.1% aqueous formic acid, gradient elution, and flow velocity is 0.3mL/min.
Wherein, described gradient elution is preferably:
During wash-out, the percent by volume of mobile phase A rises to 40% by 3% 10min consuming time, the percent by volume of Mobile phase B drops to 60% by 97% 10min consuming time accordingly, then change 100% mobile phase A into and rinse 2min, finally change mobile phase A percent by volume 3% and Mobile phase B percent by volume 97% reservation 3min into.
As preferably, described triple level Four bar Mass Spectrometer Method conditions are:
Capillary voltage: 3.5kv, taper hole voltage: 35kv, desolventizing temperature: 500 ℃, desolventizing airshed: 900L/min, taper hole blowback air flow: 30L/hr, collision cell pressure: 3.0 * 10
-3mbar; Low side resolution 1:2.5V, high-end resolution 1:15.0V, ion energy 1:0.5; Low side resolution 2:2.8V, high-end resolution 2:15.0V, ion energy 2:1.0; Ion source temperature: 150 ℃, extractor voltage: 3.0V, entrance lens voltage: 0.5V, outlet voltage: 0.5V, collision gradient: 1.0.
In addition, the present invention also provides a kind of kit of quantitative detection people beta-casein content, comprises Isotopic Internal Standard thing, the special poly saccharide peptide standard product of people's beta-casein, the special poly saccharide peptide standard product of isotope;
The polypeptide that described Isotopic Internal Standard thing is sequence as shown in SEQ ID NO:1 and from N end the valine of the 9th and the 10th 's leucine by the full isotope labeling of carbon nitrogen, the special poly saccharide peptide standard product protein sequence of the special poly saccharide peptide standard product of described people's beta-casein and isotope is the polypeptide of sequence as shown in SEQ ID NO:2, and the special poly saccharide peptide standard product sequence of the isotope valine of the 4th and the 5th 's from N end leucine is by the full isotope labeling of carbon nitrogen.
As preferably, described kit comprises Isotopic Internal Standard thing solution, the special poly saccharide peptide standard product solution of people's beta-casein, the special poly saccharide peptide standard product solution of isotope, dithiothreitol (DTT) solution, ammonium bicarbonate soln, iodo-acetamide solution, bovine trypsin solution and aqueous formic acid.
Further preferably, described bovine trypsin solution is formulated by hydrochloric acid and bovine trypsin, described bovine trypsin specific activity >3000unit/mg protein.
More preferably, each concentration of component is as follows:
The aqueous formic acid that 20 μ mol/L Isotopic Internal Standard thing solution, the special poly saccharide peptide standard product solution of 1mmol/L people's beta-casein, the special poly saccharide peptide standard product solution of 20 μ mol/L isotope, 50mmol/L dithiothreitol (DTT) solution, 50mmol/L ammonium bicarbonate soln, 150mmol/L iodo-acetamide solution, 200 μ g/mL bovine trypsin solution, percent by volume are 0.1%.
According to the method for the invention, breast milk sample to be measured is detected, quantitatively be limited to 0.12mg/100mL, day to day precision RSD is 1.01%, average recovery rate is 99.91%, matrix effect is 1.06%, be better than the 2002/657/EC of European Union instruction standard, accuracy is more accurate compared with other nonspecific detection methods.
From above technical scheme, the present invention utilizes the specific polypeptides sequence VMPVLK obtaining after people's beta-casein enzymolysis, design the sequence of isotope-labeled special peptide and isotope labeling internal standard compound, and provide a kind of brand-new accurate quantitative analysis detection method quantitatively to detect people's beta-casein based on internal standard method, there is higher accuracy, precision and sensitivity.
Embodiment
The method and the kit that the invention discloses a kind of quantitative detection people beta-casein content, those skilled in the art can use for reference content herein, suitably improve technological parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications as herein described or suitably change and combination within not departing from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: the consistance of the special peptide of isotope and the special peptide of beta-casein
The object that the present invention has introduced the special peptide of isotope is in order to overcome by extracting reagent and the caused matrix effect of matrix.In order to verify the consistance of the special peptide of the designed isotope of the present invention to the special peptide of beta-casein result under same matrix, experimental design compared the special peptide of isotope to the special peptide of beta-casein the retention time under same liquid chromatography and mass spectrum condition, daughter ion cracking mode and linearity.
Prepare respectively special peptide in the present invention and the standard series working solution of the special peptide of isotope, sample introduction analysis under identical chromatogram mass spectrum condition, obtains its retention time and equation of linear regression.Wherein the retention time of the special peptide of the beta-casein in the present invention and the special peptide of isotope is 4.75min, and both equations of linear regression have the consistance of height (to be respectively y=11435x-58588, R
2=0.9931; Y=11211x-79544, R
2=0.9921), fully prove the two consistance aspect liquid chromatography behavior.
The mass-to-charge ratio of three daughter ions of the special peptide of beta-casein is respectively 231.2m/z, 260.2m/z and 456.3m/z, and its corresponding cracking mode is respectively b2, y2 and y4.The mass-to-charge ratio of the selected daughter ion of the special peptide of isotope is respectively 231.2m/z, 267.2m/z and 469.3m/z, and first daughter ion does not comprise isotope-labeled amino acid, so its mass-to-charge ratio is compared consistent with the corresponding daughter ion of the special peptide of beta-casein; Second sub-ion packet contained a L
*therefore mass-to-charge ratio has improved 7; The 3rd daughter ion all contains V
*with L
*each, therefore its mass-to-charge ratio has improved 13.The daughter ion cracking mode of the special peptide of isotope is consistent with the cracking mode of the special peptide of beta-casein, is b2, y2 and y4.
From the above results, the special peptide of the well-designed isotope of the present invention not only can be distinguished to some extent with the special peptide of people's beta-casein on molecular weight, avoided the impact of natural isotopic abundance on this experiment, and both are at physicochemical property, chromatographic behavior, on linear equation and cracking mode, can be consistent, meet the requirement of the internal target character of experiment.
Embodiment 2: the contrast of Isotopic Internal Standard thing and people's beta-casein enzymolysis efficiency
In order to verify whether Isotopic Internal Standard thing and people's beta-casein in the present invention all have more close enzymolysis efficiency in various matrix, following test has been carried out in design:
By ammonium bicarbonate soln 1:9 dilution for breast milk, precision measures breast milk after 10 μ L dilutions in reaction tube, in reaction tube, add respectively the skimmed milk solution (1g skimmed milk powder is dissolved in 100mL50mM ammonium bicarbonate soln) of 0,5,20,50,200 μ L as matrix, this matrix can be simulated the various nutritional labelings in breast milk, and on not impact of quantitative detection.Adding subsequently ammonium bicarbonate soln to cumulative volume is 945 μ L, add 10 μ L Isotopic Internal Standard thing solution and 10 μ L dithiothreitol (DTT) solution, after shaking up in 60 ℃ of isothermal reaction 30min, taking-up is cooled to room temperature, add 10 μ L iodo-acetamide solution, the standing 30min in dark place at room temperature, add 10 μ L bovine trypsin solution, after 37 ℃ of constant temperature enzymolysis 2h, add the aqueous formic acid that 5 μ L percents by volume are 0.1%, the standing 1h of room temperature, finally by the sample introduction analysis after 0.22 μ m filtering with microporous membrane of gained solution.
Result shows, the relative standard deviation of the peak area of the special peptide of both beta-caseins is 5.71%, the relative standard deviation of the special peptide of beta-casein and the special peptide peak area ratio of isotope is 3.27%, illustrate that the method for the invention has the enzymolysis efficiency same with people's beta-casein, can obtain the special peptide of isotope of equivalent, can effectively get rid of the impact that matrix effect brings enzymolysis and mass spectrum ionization, the people's beta-casein in sample is done to accurate quantitative analysis.
Embodiment 3: the preparation of reagent
1, the preparation of the special poly saccharide peptide standard product solution of beta-casein: accurately pipette 1mL ultrapure water, add in the pipe of the special peptide of beta-casein accurately weighing in advance, ultrasonic dissolution 30s, gained solution is the special poly saccharide peptide standard product solution of beta-casein of 1mmol/L;
2, the preparation of the special poly saccharide peptide standard product solution of isotope: accurately pipette 1mL ultrapure water, add in the pipe of the special peptide of isotope accurately weighing in advance, ultrasonic dissolution 30s, gained solution is the special poly saccharide peptide standard product solution of isotope of 20 μ mol/L;
3, the preparation of Isotopic Internal Standard thing solution: accurately pipette 1mL ultrapure water, add in the pipe of the Isotopic Internal Standard thing accurately weighing in advance, ultrasonic dissolution 30s, gained solution is the Isotopic Internal Standard thing solution of 20 μ mol/L;
4, the preparation of bovine trypsin solution: accurately pipette 1mL 1mmol/L hydrochloric acid solution, add in the pipe of the bovine trypsin accurately weighing in advance, (specific activity >3000unit/mg protein), ultrasonic dissolution 30s, gained solution is the trypsin solution of 200 μ g/mL;
5, ammonium bicarbonate (NH
4hCO
3) preparation of solution: accurately take 1.98g NH
4hCO
3in 500mL volumetric flask, add ultrapure water ultrasonic dissolution 3min, to be cooled to constant volume after room temperature to scale, gained solution is the ammonium bicarbonate soln of 50mmol/L;
6, the preparation of iodo-acetamide (IAA) solution: accurately take 277mg IAA in 10mL volumetric flask, the about 9mL of ammonium bicarbonate soln that adds 50mmol/L, ultrasonic dissolution 3min, to be cooled to constant volume after room temperature to scale, gained solution is the iodo-acetamide solution of 150mmol/L;
7, the preparation of dithiothreitol (DTT) (DTT) solution: accurately take 77mg DTT in 10mL volumetric flask, the about 9mL of ammonium bicarbonate soln that adds 50mmol/L, ultrasonic dissolution 3min, to be cooled to constant volume after room temperature to scale, gained solution is the dithiothreitol (DTT) solution of 50mmol/L.
Embodiment 4: the method for the invention
Liquid chromatography: Acquity Ultra Performance LC(Waters company, the U.S.)
Triple level Four bar mass spectrums: Xevo TQ MS(Waters company, the U.S.)
High performance liquid chromatography separation condition:
Chromatographic column is C18 chromatographic column, and column temperature is 40 ℃; Mobile phase A is that percent by volume is the acetonitrile solution of 0.1% formic acid, and Mobile phase B is that percent by volume is 0.1% aqueous formic acid, gradient elution, and flow velocity is 0.3mL/min.
Wherein, gradient elution is:
During wash-out, the percent by volume of mobile phase A rises to 40% by 3% 10min consuming time, the percent by volume of Mobile phase B drops to 60% by 97% 10min consuming time accordingly, then change 100% mobile phase A into and rinse 2min, finally change mobile phase A percent by volume 3% and Mobile phase B percent by volume 97% reservation 3min into.
Triple level Four bar Mass Spectrometer Method conditions are:
Capillary voltage: 3.5kv, taper hole voltage: 35kv, desolventizing temperature: 500 ℃, desolventizing airshed: 900L/min, taper hole blowback air flow: 30L/hr, collision cell pressure: 3.0 * 10
-3mbar; Low side resolution 1:2.5V, high-end resolution 1:15.0V, ion energy 1:0.5; Low side resolution 2:2.8V, high-end resolution 2:15.0V, ion energy 2:1.0; Ion source temperature: 150 ℃, extractor voltage: 3.0V, entrance lens voltage: 0.5V, outlet voltage: 0.5V, collision gradient: 1.0.
2, method
By 50mmol/L ammonium bicarbonate soln 1:9 dilution by volume for breast milk sample to be measured, get 10 μ L diluted breast milk samples, then add 20 μ mol/L Isotopic Internal Standard thing solution 10 μ L, 50mmol/L dithiothreitol (DTT) solution 10 μ L and 50mmol/L ammonium bicarbonate soln 945 μ L, after shaking up in 60 ℃ of isothermal reaction 30min, taking-up is cooled to room temperature, add the 150mmol/L iodo-acetamide solution 10 standing 30min in μ L dark place, then add 200 μ g/mL bovine trypsin solution 10 μ L, after 37 ℃ of constant temperature enzymolysis 2h, add the pure formic acid of 5 μ L, the standing 1h of room temperature, finally by gained solution after 0.22 μ m filtering with microporous membrane, by high performance liquid chromatography triple level Four bar Mass Spectrometer Method of connecting, with internal standard method, obtain the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope,
Get respectively the special peptide solution 1 μ L of 1mmol/L people's beta-casein, 10 μ L, 20 μ L, 30 μ L, 40 μ L and 50 μ L, add 50mmol/L ammonium bicarbonate soln to 1mL.
Get each 10 μ L of the special poly saccharide peptide standard product solution of people's beta-casein of above-mentioned gradient concentration, after adding respectively the special poly saccharide peptide standard product solution 10 μ L of 20 μ mol/L isotope, with 50mmol/L ammonium bicarbonate soln 975 μ L, dilute, finally add the aqueous formic acid that 5 μ L percents by volume are 0.1%, the standing 1h of room temperature, finally by gained solution after 0.22 μ m filtering with microporous membrane, by high performance liquid chromatography triple level Four bar Mass Spectrometer Method of connecting, the typical curve of the peak area ratio that obtains the special peptide of people's beta-casein and the special peptide of isotope and the corresponding special poly saccharide peptide standard product solution concentration of people's beta-casein.
According to the typical curve obtaining, carry out linear regression, draw linear equation Y=kX+b, wherein Y is the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope; X is the volumetric molar concentration of the special peptide of people's beta-casein, and unit is nmol/L; K is the slope of linear equation; B is the intercept of linear equation.By the peak area ratio substitution linear equation of the aforementioned special peptide of people's beta-casein obtaining and the special peptide of isotope, can calculate the special peptide volumetric molar concentration of people's beta-casein in breast milk sample to be measured.
By the special peptide concentration substitution of the people's beta-casein cubage formula in breast milk sample to be measured: the special peptide volumetric molar concentration * people beta-casein of beta-casein molal weight * 10 in people's beta-casein content=breast milk sample
3, can obtain the content of people's beta-casein in tested breast milk sample.
Embodiment 5: the method for the invention day to day precision detects
Select the breast milk sample in same source to carry out repeated detection in different number of days different time sections, the results are shown in Table 1.
Table 1 day to day precision testing result
As shown in Table 1, day to day precision RSD of the present invention is only 1.01%, meet the requirement of the 2002/657/EC of European Union instruction to precision <5.64%, and far below this standard, precision is more excellent.
Embodiment 6: the method for the invention quantitative limit detects
Select the special peptide solution sample introduction of 2nmol/L to analyze, obtain the signal to noise ratio (S/N ratio) of chromatographic peak.The 10 times of signal to noise ratio (S/N ratio)s of take are quantitative limit, take embodiment 4 methods as basis, the special peptide volumetric molar concentration * people beta-casein of beta-casein molal weight * 10 in substitution formula beta-casein detection limit=breast milk sample to be measured
3÷ signal to noise ratio (S/N ratio) * 10, calculate this method and be quantitatively limited to 0.12mg/100mL, in measuring the process of quantitative limit, too low or while reaching quantitative detecting method quantitative limit critical point of the present invention in the special peptide volumetric molar concentration of people's ALA, detection is vulnerable to disturb and causes signal to noise ratio (S/N ratio) fluctuation, therefore for the purpose of rigorous, the present embodiment has carried out revision test 3 times, get mxm. as the quantitative limit of quantitative detecting method of the present invention, concrete outcome is in Table 2.
Table 2 quantitative limit testing result
| Parallel sample | Signal to noise ratio (S/N ratio) | Method quantitative limit [mg/100mL] |
| 1 | 744 | 0.06 |
| 2 | 411 | 0.12 |
| 3 | 718 | 0.07 |
Embodiment 7: the detection of the method for the invention recovery
By 50mmol/L ammonium bicarbonate soln 1:9 dilution by volume for breast milk sample to be measured, get 10 μ L diluted breast milk samples, then add 20 μ mol/L Isotopic Internal Standard thing solution 10 μ L, 10 μ mol/L(low concentration mark-ons), concentration mark-on in 20 μ mol/L() or 30 μ mol/L(high concentration mark-ons) the special peptide solution 10 μ L of beta-casein, 50mmol/L dithiothreitol (DTT) solution 10 μ L and 50mmol/L ammonium bicarbonate soln 935 μ L, after shaking up in 60 ℃ of isothermal reaction 30min, taking-up is cooled to room temperature, add the 150mmol/L iodo-acetamide solution 10 standing 30min in μ L dark place, then add 200 μ g/mL bovine trypsin solution 10 μ L, after 37 ℃ of constant temperature enzymolysis 2h, to add 5 μ L percents by volume be 0.1% adds aqueous acid, the standing 1h of room temperature, finally by gained solution after 0.22 μ m filtering with microporous membrane, by high performance liquid chromatography triple level Four bar Mass Spectrometer Method of connecting, with internal standard method, obtain the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope, calculate the concentration of special peptide, thereby extrapolate the recovery.
Table 3 recovery statistics
As shown in Table 3, the recovery of the present invention is 99.91%, meets the requirement of the 2002/657/EC of European Union instruction to recovery 95-105%, and data approach 100%, and the recovery is better.
Embodiment 8: the detection of the method for the invention matrix effect.
Get respectively the special peptide solution 1 μ L of 1mmol/L people's beta-casein, 10 μ L, 20 μ L, 30 μ L, 40 μ L and 50 μ L, add 50mmol/L ammonium bicarbonate soln to 1mL.
Get each 10 μ L of the special poly saccharide peptide standard product solution of beta-casein of above-mentioned gradient concentration, after adding respectively the special poly saccharide peptide standard product solution 10 μ L of 20 μ mol/L isotope, with 50mmol/L ammonium bicarbonate soln 975 μ L, dilute, finally add the aqueous formic acid that 5 μ L percents by volume are 0.1%, by high performance liquid chromatography triple level Four bar Mass Spectrometer Method of connecting, the typical curve of the peak area ratio that obtains the special peptide of people's beta-casein and the special peptide of isotope and the corresponding special poly saccharide peptide standard product solution concentration of beta-casein.
Get each 10 μ L of the special poly saccharide peptide standard product solution of beta-casein of above-mentioned gradient concentration, add respectively the special poly saccharide peptide standard product solution 10 μ L of 20 μ mol/L isotope, breast milk solution after dilution (50mmol/L ammonium bicarbonate soln 1:9 dilution by volume for breast milk sample to be measured) 10 μ L, 50mmol/L dithiothreitol (DTT) solution 10 μ L and 50mmol/L ammonium bicarbonate soln 945 μ L react, after reaction, add 150mmol/L iodo-acetamide solution 10 μ L dark places standing, add the aqueous formic acid that 5 μ L percents by volume are 1%, by high performance liquid chromatography triple level Four bar Mass Spectrometer Method of connecting, the matrix curve of the peak area ratio that obtains the special peptide of people's beta-casein and the special peptide of isotope and the corresponding special poly saccharide peptide standard product solution concentration of people's beta-casein.
Matrix effect can obtain by standard of comparison curve and matrix slope of a curve, and concrete outcome is referring to table 4.
The testing result of table 4 matrix effect
| Parallel sample | Slope of standard curve | Matrix rate of curve | Matrix effect |
| 1 | 0.773191 | 0.786551 | 1.71% |
| 2 | 0.779535 | 0.772594 | 0.89% |
| 3 | 0.782266 | 0.78687 | 0.59% |
| ? | ? | Mean value | 1.06% |
As shown in Table 4, matrix effect of the present invention is 1.06%, meet the requirement of the 2002/657/EC of European Union instruction to matrix effect <10%, and data is far below standard-required, show that the method for the invention accuracy is higher, be subject to the impact of matrix effect extremely low.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. quantitatively detect a method for people's beta-casein content, it is characterized in that, comprise the steps:
Step 1, by 50mmol/L ammonium bicarbonate soln dilution for breast milk sample to be measured, get 10 μ L diluted breast milk samples, then add 20 μ mol/L Isotopic Internal Standard thing solution 10 μ L, 50mmol/L dithiothreitol (DTT) solution 10 μ L and 50mmol/L ammonium bicarbonate soln 945 μ L react, after reaction, add 150mmol/L iodo-acetamide solution 10 μ L dark places standing, then add 200 μ g/mL bovine trypsin solution 10 μ L enzymolysis, add after the pure formic acid of 5 μ L by high performance liquid chromatography triple level Four bar Mass Spectrometer Method of connecting, with internal standard method, obtain the peak area ratio of the special peptide of people's beta-casein and the special peptide of isotope,
Step 2, get each 10 μ L of the special poly saccharide peptide standard product solution of people's beta-casein of gradient concentration, after adding respectively the special poly saccharide peptide standard product solution 10 μ L of 20 μ mol/L isotope, with 50mmol/L ammonium bicarbonate soln 975 μ L, dilute, finally add the aqueous formic acid that 5 μ L percents by volume are 0.1%, by high performance liquid chromatography triple level Four bar Mass Spectrometer Method of connecting, the typical curve of the peak area ratio that obtains the special peptide of people's beta-casein and the special peptide of isotope and the corresponding special poly saccharide peptide standard product solution concentration of people's beta-casein, the special peptide of people's beta-casein that step 1 is obtained and the peak area ratio substitution typical curve of the special peptide of isotope obtain the concentration of the special peptide of beta-casein in breast milk sample, then be calculated as follows and obtain people's beta-casein content,
The special peptide volumetric molar concentration * people of beta-casein beta-casein molal weight * breast milk Sample Dilution multiple in people's beta-casein content=breast milk sample;
Wherein, the polypeptide that described Isotopic Internal Standard thing is sequence as shown in SEQ ID NO:1 and from N end the valine of the 9th and the 10th 's leucine by the full isotope labeling of carbon nitrogen, the special poly saccharide peptide standard product protein sequence of the special poly saccharide peptide standard product of described people's beta-casein and isotope is the polypeptide of sequence as shown in SEQ ID NO:2, and the special poly saccharide peptide standard product sequence of the isotope valine of the 4th and the 5th 's from N end leucine is by the full isotope labeling of carbon nitrogen.
2. method according to claim 1, is characterized in that, 50mmol/L ammonium bicarbonate soln 1:9 dilution by volume for described breast milk sample to be measured.
3. method according to claim 1, is characterized in that, described bovine trypsin solution is formulated by hydrochloric acid and bovine trypsin, described bovine trypsin specific activity >3000unit/mg protein.
4. method according to claim 1, is characterized in that, described enzymolysis is at 37 ℃ of constant temperature enzymolysis 2h.
5. method according to claim 1, is characterized in that, the special poly saccharide peptide standard product solution of beta-casein of described gradient concentration is prepared in accordance with the following methods:
Get respectively the special peptide solution 1 μ L of 1mmol/L beta-casein, 10 μ L, 20 μ L, 30 μ L, 40 μ L and 50 μ L, add ammonium bicarbonate soln to 1mL.
6. method according to claim 1, is characterized in that described high performance liquid chromatography separation condition:
Chromatographic column is C18 chromatographic column, and column temperature is 40 ℃; Mobile phase A is that percent by volume is the acetonitrile solution of 0.1% formic acid, and Mobile phase B is that percent by volume is 0.1% aqueous formic acid, gradient elution, and flow velocity is 0.3mL/min.
7. method according to claim 1, is characterized in that, described triple level Four bar Mass Spectrometer Method conditions are:
Capillary voltage: 3.5kv, taper hole voltage: 35kv, desolventizing temperature: 500 ℃, desolventizing airshed: 900L/min, taper hole blowback air flow: 30L/hr, collision cell pressure: 3.0 * 10
-3mbar; Low side resolution 1:2.5V, high-end resolution 1:15.0V, ion energy 1:0.5; Low side resolution 2:2.8V, high-end resolution 2:15.0V, ion energy 2:1.0; Ion source temperature: 150 ℃, extractor voltage: 3.0V, entrance lens voltage: 0.5V, outlet voltage: 0.5V, collision gradient: 1.0.
8. quantitatively detect a kit for people's beta-casein content, it is characterized in that, comprise Isotopic Internal Standard thing, the special poly saccharide peptide standard product of people's beta-casein, the special poly saccharide peptide standard product of isotope;
The polypeptide that described Isotopic Internal Standard thing is sequence as shown in SEQ ID NO:1 and from N end the valine of the 9th and the 10th 's leucine by the full isotope labeling of carbon nitrogen, the special poly saccharide peptide standard product protein sequence of the special poly saccharide peptide standard product of described people's beta-casein and isotope is the polypeptide of sequence as shown in SEQ ID NO:2, and the special poly saccharide peptide standard product sequence of the isotope valine of the 4th and the 5th 's from N end leucine is by the full isotope labeling of carbon nitrogen.
9. kit according to claim 8, it is characterized in that, comprise Isotopic Internal Standard thing solution, the special poly saccharide peptide standard product solution of people's beta-casein, the special poly saccharide peptide standard product solution of isotope, dithiothreitol (DTT) solution, ammonium bicarbonate soln, iodo-acetamide solution, bovine trypsin solution and aqueous formic acid.
10. kit according to claim 9, is characterized in that, each concentration of component is as follows:
The aqueous formic acid that 20 μ mol/L Isotopic Internal Standard thing solution, the special poly saccharide peptide standard product solution of 1mmol/L people's beta-casein, the special poly saccharide peptide standard product solution of 20 μ mol/L isotope, 50mmol/L dithiothreitol (DTT) solution, 50mmol/L ammonium bicarbonate soln, 150mmol/L iodo-acetamide solution, 200 μ g/mL bovine trypsin solution, percent by volume are 0.1%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310656063.8A CN103616454B (en) | 2013-12-06 | 2013-12-06 | Method and kit for quantitatively detecting human beta-casein content |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310656063.8A CN103616454B (en) | 2013-12-06 | 2013-12-06 | Method and kit for quantitatively detecting human beta-casein content |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103616454A true CN103616454A (en) | 2014-03-05 |
| CN103616454B CN103616454B (en) | 2015-06-03 |
Family
ID=50167160
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310656063.8A Active CN103616454B (en) | 2013-12-06 | 2013-12-06 | Method and kit for quantitatively detecting human beta-casein content |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103616454B (en) |
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645464A (en) * | 2015-12-17 | 2017-05-10 | 中国医科大学 | Kit and detection method for realizing bovine beta-casein identification and absolute quantification |
| CN106749598A (en) * | 2016-11-30 | 2017-05-31 | 杭州帕匹德科技有限公司 | A kind of feature peptide for detecting the adulterated ratio of milk powder in goat milk powder is combined and method |
| CN106980021A (en) * | 2017-03-31 | 2017-07-25 | 李森康 | A kind of protein detection techniques based on enzymolysis polypeptide principle for being able to verify that hydrolysis result |
| CN107037173A (en) * | 2017-03-31 | 2017-08-11 | 李森康 | A kind of method of protein content in quantitatively detection cattle and sheep breast |
| CN108152385A (en) * | 2017-11-10 | 2018-06-12 | 杭州谱胜检测科技有限责任公司 | The kit of the detection method of protein content of whey and realization this method in a kind of baby formula milk powder |
| CN108519485A (en) * | 2018-04-10 | 2018-09-11 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | A kind of Mass Spectrometry detection method of A1/A2 beta-caseins |
| CN108709939A (en) * | 2018-05-21 | 2018-10-26 | 杭州璞湃科技有限公司 | A kind of feature peptide and method for detecting A2 beta-casein contents in cow's milk product |
| CN108732253A (en) * | 2017-04-14 | 2018-11-02 | 杭州量康科技有限公司 | Peptide fragment composition and assay method for serous proteinquatative measurement |
| CN109444287A (en) * | 2018-12-20 | 2019-03-08 | 杭州同创医学检验实验室有限公司 | A kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein |
| CN109917032A (en) * | 2019-03-13 | 2019-06-21 | 杭州老爸评测科技有限公司 | A kind of quantitative detecting method turning Bt albumen in Bt protein food |
| CN114137114A (en) * | 2020-11-26 | 2022-03-04 | 信立泰(苏州)药业有限公司 | Teriparatide isotope internal standard, preparation method and application thereof |
| EP4105654A4 (en) * | 2021-08-06 | 2023-10-18 | Beijing Sanyuan Foods Co., Ltd. | Method for measuring content of whey protein and content of casein in powdered milk and/or ratio of two |
| CN117347531A (en) * | 2023-12-05 | 2024-01-05 | 天津医科大学总医院 | Method for detecting protein by adopting double internal calibration quantity |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1691961A (en) * | 2002-11-29 | 2005-11-02 | 森永乳业株式会社 | Cysteine protease inhibitor |
| WO2006131586A1 (en) * | 2005-06-08 | 2006-12-14 | Consejo Superior De Investigaciones Científicas | Bioactive peptides identified in enzymatic hydrolyzates of milk caseins and method of obtaining same |
| CN102590413A (en) * | 2012-01-18 | 2012-07-18 | 浙江省疾病预防控制中心 | Quantitative detection method for bovine alpha-lactalbumin |
| CN103134881A (en) * | 2013-02-01 | 2013-06-05 | 浙江省疾病预防控制中心 | Cattle 2, 2-bipyridine-lactalbumin quantitative detection reagent box and application thereof |
| CN103293317A (en) * | 2013-04-28 | 2013-09-11 | 浙江省疾病预防控制中心 | Cow milk beta-lactoglobulin quantitative determination kit and application thereof |
-
2013
- 2013-12-06 CN CN201310656063.8A patent/CN103616454B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1691961A (en) * | 2002-11-29 | 2005-11-02 | 森永乳业株式会社 | Cysteine protease inhibitor |
| WO2006131586A1 (en) * | 2005-06-08 | 2006-12-14 | Consejo Superior De Investigaciones Científicas | Bioactive peptides identified in enzymatic hydrolyzates of milk caseins and method of obtaining same |
| CN102590413A (en) * | 2012-01-18 | 2012-07-18 | 浙江省疾病预防控制中心 | Quantitative detection method for bovine alpha-lactalbumin |
| CN103134881A (en) * | 2013-02-01 | 2013-06-05 | 浙江省疾病预防控制中心 | Cattle 2, 2-bipyridine-lactalbumin quantitative detection reagent box and application thereof |
| CN103293317A (en) * | 2013-04-28 | 2013-09-11 | 浙江省疾病预防控制中心 | Cow milk beta-lactoglobulin quantitative determination kit and application thereof |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645464A (en) * | 2015-12-17 | 2017-05-10 | 中国医科大学 | Kit and detection method for realizing bovine beta-casein identification and absolute quantification |
| CN106770812A (en) * | 2015-12-17 | 2017-05-31 | 中国医科大学 | It is capable of achieving ox ɑs1Casein identification and the kit and assay method of absolute quantitation |
| CN106749598A (en) * | 2016-11-30 | 2017-05-31 | 杭州帕匹德科技有限公司 | A kind of feature peptide for detecting the adulterated ratio of milk powder in goat milk powder is combined and method |
| CN106980021A (en) * | 2017-03-31 | 2017-07-25 | 李森康 | A kind of protein detection techniques based on enzymolysis polypeptide principle for being able to verify that hydrolysis result |
| CN107037173A (en) * | 2017-03-31 | 2017-08-11 | 李森康 | A kind of method of protein content in quantitatively detection cattle and sheep breast |
| CN108732253A (en) * | 2017-04-14 | 2018-11-02 | 杭州量康科技有限公司 | Peptide fragment composition and assay method for serous proteinquatative measurement |
| CN108152385A (en) * | 2017-11-10 | 2018-06-12 | 杭州谱胜检测科技有限责任公司 | The kit of the detection method of protein content of whey and realization this method in a kind of baby formula milk powder |
| CN108519485A (en) * | 2018-04-10 | 2018-09-11 | 上海出入境检验检疫局动植物与食品检验检疫技术中心 | A kind of Mass Spectrometry detection method of A1/A2 beta-caseins |
| CN108709939A (en) * | 2018-05-21 | 2018-10-26 | 杭州璞湃科技有限公司 | A kind of feature peptide and method for detecting A2 beta-casein contents in cow's milk product |
| CN109444287A (en) * | 2018-12-20 | 2019-03-08 | 杭州同创医学检验实验室有限公司 | A kind of LC-MS/MS quantitative detecting method of alpha-fetoprotein |
| CN109444287B (en) * | 2018-12-20 | 2019-12-17 | 杭州同创医学检验实验室有限公司 | LC-MS/MS quantitative detection method for alpha fetoprotein |
| CN109917032A (en) * | 2019-03-13 | 2019-06-21 | 杭州老爸评测科技有限公司 | A kind of quantitative detecting method turning Bt albumen in Bt protein food |
| CN109917032B (en) * | 2019-03-13 | 2021-08-24 | 杭州老爸评测科技有限公司 | Quantitative detection method for Bt protein in Bt protein-transgenic food |
| CN114137114A (en) * | 2020-11-26 | 2022-03-04 | 信立泰(苏州)药业有限公司 | Teriparatide isotope internal standard, preparation method and application thereof |
| EP4105654A4 (en) * | 2021-08-06 | 2023-10-18 | Beijing Sanyuan Foods Co., Ltd. | Method for measuring content of whey protein and content of casein in powdered milk and/or ratio of two |
| CN117347531A (en) * | 2023-12-05 | 2024-01-05 | 天津医科大学总医院 | Method for detecting protein by adopting double internal calibration quantity |
| CN117347531B (en) * | 2023-12-05 | 2024-02-13 | 天津医科大学总医院 | Method for detecting protein by adopting double internal calibration quantity |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103616454B (en) | 2015-06-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103616454B (en) | Method and kit for quantitatively detecting human beta-casein content | |
| CN103642895B (en) | A kind of method of detection by quantitative people ALA content and test kit | |
| Delafield et al. | Recent advances in analytical approaches for glycan and glycopeptide quantitation | |
| Bąchor et al. | Trends in the design of new isobaric labeling reagents for quantitative proteomics | |
| CN104614479B (en) | A kind of detection method of food vitamins | |
| CN103529138B (en) | Bovine beta-casein quantitative determination kit and application thereof | |
| EP2098859A1 (en) | Method for quantification of peptide and protein | |
| CN101910819A (en) | Detect the method for dihydroxyvitamin D metabolites by mass spectroscopy | |
| CN103293317B (en) | A kind of cow's milk beta lactoglobulin immue quantitative detection reagent box and application thereof | |
| CN110850099B (en) | Method for valuing C-reactive protein in serum for non-disease diagnosis | |
| CN104345107B (en) | Lac Bovis seu Bubali serum albumin immue quantitative detection reagent box in a kind of breast or milk product | |
| WO2002084250A2 (en) | Methods for mass spectrometry detection and quantification of specific target proteins in complex biological samples | |
| CN103278574B (en) | Method for detecting hemoglobin by liquid chromatogram-triple tandem quadrupole mass spectrometer | |
| CN106749598A (en) | A kind of feature peptide for detecting the adulterated ratio of milk powder in goat milk powder is combined and method | |
| CN1979155A (en) | High-sensitive blood-plasma total homocysteine detection reagent box | |
| CN102435680B (en) | Bovine serum albumin non-labeled mass spectrum qualitative and quantitative detection method | |
| CN103134881A (en) | Cattle 2, 2-bipyridine-lactalbumin quantitative detection reagent box and application thereof | |
| CN103134860A (en) | Quantitative determination method for target peptides and proteins | |
| Wither et al. | Mass spectrometry‐based bottom‐up proteomics: Sample preparation, LC‐MS/MS analysis, and database query strategies | |
| CN106749600A (en) | A kind of labelled peptide of CPP and its application | |
| CN117230137A (en) | Method for absolute quantification of low abundance polypeptides using mass spectrometry | |
| CN104122339A (en) | Isotopic abundance detection method for D, 13C or 15N labeled organic compounds | |
| CN108548876A (en) | The identification of phosphated peptide section and quantitative approach in a kind of improved biological sample | |
| CN105445409A (en) | Method for measuring glycosylated hemoglobin by isotope dilution mass spectrometry | |
| CN109030638A (en) | A method of for measuring selenomethionine in bio-matrix |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C53 | Correction of patent of invention or patent application | ||
| CB02 | Change of applicant information |
Address after: 310051 No. 3758 South Ring Road, Hangzhou, Zhejiang, Binjiang District Applicant after: Bei Yinmei Ying Tong food limited-liability company Address before: 310051 No. 3758 South Ring Road, Hangzhou, Zhejiang, Binjiang District Applicant before: Zhejiang Beingmate Group Co., Ltd. |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: APPLICANT; FROM: ZHEJIANG BEINGMATE GROUP CO., LTD. TO: BEINGMATE BABY FOOD CO., LTD. |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190505 Address after: 310052 19, South Ring Road, Binjiang District, Hangzhou, Zhejiang, 19 Patentee after: Beingmate (Hangzhou) Food Research Institute Co., Ltd. Address before: 310051 No. 3758 South Ring Road, Binjiang District, Hangzhou City, Zhejiang Province Patentee before: Bei Yinmei Ying Tong food limited-liability company |