CN101339159B - Milk alpha-casein content checking method - Google Patents
Milk alpha-casein content checking method Download PDFInfo
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Abstract
The invention relates to a detection method for the content of Alpha-casein in emulsion, comprising the following steps: an emulsion sample which needs detection and sample buffer solution are done with mixed processing and the processed sample is done with detection by a capillary electrophoresis apparatus; the preparation method of the sample buffer solution comprises the following steps: trimethyl aminomethane buffer is added with 3-morpholinopropane sulphonic acid, ethylene diaminetetraacetic acid disodium and urea; when the sample is processed, Beta-mercaptoethanol and methyl hydroxyethyl cellulose are added; wherein, the processing steps of the emulsion sample comprises: centrifuge processing of the emulsion sample, taking the supernatant liquid of the processed sample to be mixed with the sample buffer solution and then washed by ultrasonic wave; the method of the invention has good separating effect and fast speed and can use an uncoated capillary column; the related coefficient of an equation of linear regression of the method of the invention is more than 0.98, the lowest detection line is 0.15ng, the lowest detection concentration is 0.005mg/ml and the precision is up to more than 90 percent.
Description
Technical field
The present invention relates to the detection method of a kind of Ruzhong alpha-casein content; Especially a kind of method of utilizing Capillary Electrophoresis to detect the Ruzhong alpha-casein content particularly detects the improvement of the treatment step of sample in the alpha-casein content process of Ruzhong about Capillary Electrophoresis.
Background technology
Casilan is one of major product of dairy industry, because it has excellent functional properties and higher nutritive value, therefore in food industry and other industry, has all obtained using widely.Casein has higher nutritive value, can add in other food and strengthen protein.Casein can form colloid in water, have certain viscosity and good water retention characteristic, in icecream production.Add casein sodium and make stabilizing agent, have the fatty emulsification of enhancing, preserve moisture and improve effects such as viscosity.Exist tangible hydrophobic region and hydrophilic area in the casein, therefore have emulsibility and foaminess preferably.In recent years; Increasing to casein physiological function and bioactive research; Adopt the whole bag of tricks modification to prepare modified product to casein and also more and more receive publicity, so the production of casein modified product will become a megatrend of development of food industry from now on.Therefore accurately detecting the Ruzhong alpha-casein content has very important significance for the proportioning of various functions and nutrition in the Dairy Processing process.
Alpha-casein accounts for the 45-55% of the total casein content of cow's milk greatly, is the element in the casein glue kernel structure, mainly contains α s1-casein and two kinds of forms of α s2-casein.Alpha-casein is made up of 199 amino acid, combines 8 phosphate anions on each molecule, and binding site mainly concentrates on the 43-79 position of peptide chain, can obtain phosphopeptide at this position through enzymolysis.
At present, domestic national standard and the industry standard that the alpha-casein content detection method is not arranged as yet.Relevant state indicates DIN-10466-2001 " mensuration of protein content of whey in whole albumen of milk and milk product ", DIN-10470-1999 " content of milk protein and casein content are measured in all protein of milk and milk product ", DIN-10472-1996 " lactoprotein and casein composition quantitative determination-electrophoresis in milk and the dairy products total protein concentration ".
HPCE is that one type of liquid phase differential that comprises electrophoresis, chromatogram and intersection content thereof is from technology.It can accurately detect the functional component of mingling composition, microbiotic, interpolation, lactalbumin, immunoglobulin (Ig), enzyme, sugar, adjuvant and other micromolecular content in the milk fast.HPCE detects least concentration can reach 10.5-10.8mol/L.This method is easy and simple to handle, accurate, and detecting out as a result, the time is merely about 1-2h.At present, the application prospect of HPCE is very extensive.
" application of context of detection is measured and mingled to Capillary Electrophoresis casein content in cow's milk " (" food and fermentation industries "; 2005 the 31st the 1st phases of volume; Open that east is given, Pang Guangchang, high France, Zhang Tao, Chen Qingsen, Hu Zhi with) disclose a kind of capillary electrophoresis and separate and detect caseic method in the raw milk; This method centrifuge stock breast comes fractionation of fatty, but the time of centrifugal 30min is longer, causes that easily Partial Protein deposition or fat are residual.In addition, this method also discloses a kind of sample buffer solution, yet it is still not ideal enough to caseic separating effect.
" capillary electrophoresis of protein determination research in the dairy produce "; (" Journal of Analytical Science "; In February, 2006, the 22nd the 1st phase of volume, Tang Ping, Tian Jing, She Zhenbao, father-in-law forward, Xu Guowang) also disclose a kind of use capillary electrophoresis and detected caseic quantitative analysis method in the dairy produce.
Above-mentioned two kinds of methods have all been used long capillary column having coated layer, but long capillary column disengaging time is also longer, higher relatively, the easy damage of the price of capillary column having coated layer, harsh to the environment for use requirement.
Summary of the invention
The technical matters that the present invention will solve provides a kind of method of utilizing Capillary Electrophoresis to detect the Ruzhong alpha-casein content, to improve the separating effect of alpha-casein, shortens detection time, reduces and detects cost.
Another technical matters that the present invention will solve provides the detection method of a kind of Ruzhong alpha-casein content, so that a kind of improved sample preparation step to be provided.
Another technical matters that the present invention will solve is to detect cost through adopting short not capillary column having coated layer, reducing, and improves detection speed.
For solving the problems of the technologies described above; The invention provides the detection method of a kind of Ruzhong alpha-casein content; This method may further comprise the steps: the newborn sample and the sample buffer that will detect carry out hybrid processing; Sample after the hybrid processing is detected through HPCE; The compound method of said sample buffer is: the trimethyl aminomethane buffer solution adds 3-morpholino propane sulfonic acid, disodium ethylene diamine tetraacetate, urea, adds beta-mercaptoethanol, methyl hydroxyethylcellulose when handling sample again.
Preferably; The compound method of sample buffer is: 160mmol/L trimethyl aminomethane buffer solution adds 40mmol/L3-morpholino propane sulfonic acid, 60mmol/L disodium ethylene diamine tetraacetate, 7mol/L urea; Add the beta-mercaptoethanol of 5ul/ml when handling sample again; Methyl hydroxyethylcellulose 0.05% is regulated pH value to 8.5.
Preferably, with before said sample buffer mixes, newborn sample is carried out centrifugal treating at said newborn sample, centrifugal condition is: 2000-6000r/min, centrifugal 5-15min, preferred 4500r/min, 10min.Centrifugal purpose is to remove fat, avoids lipochondrion to separating the influence of peak value and disengaging time.
Preferably, before the sample after the said hybrid processing being detected through said HPCE, with the sample after the said hybrid processing with ultrasonic cleaning 1-10min.Ultrasonic cleaning can prevent that gas is arranged in the damping fluid, and containing gas in the damping fluid will influence electric current, make it fluctuated, influence testing result.
Preferably; Said detection method further comprises the steps: 1) alpha-casein is dissolved in the said sample buffer; Prepare the standard solution of a plurality of different content alpha-caseins, and said a plurality of standard solution are detected the typical curve that obtains between alpha-casein content and the testing result respectively through said HPCE; 2) will detect said newborn test result of samples and the comparison of said typical curve that obtains through said HPCE, obtain the content of alpha-casein in the said newborn sample.
Above-mentioned steps 1) in, in said sample buffer, dissolves the alpha-casein standard substance, be mixed with the standard solution of 4mg/ml, 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml.
The supernatant of said newborn sample or said alpha-casein standard substance and said sample buffer use eddy mixer to shake up.This instrument ability biased sample is more even, can not produce any destruction to sample simultaneously.
Above-mentioned HPCE detected parameters is following: detected temperatures 15-40 ℃; Capillary column diameter 25-75 μ m, length 20-600mm, PDAD or UV-detector, pressure sample introduction or electrokinetic injection; Pressure is 0-1psi; Sample injection time 1s-20s, detected temperatures is 15-40 ℃, WV 7-30kV; Preferred parameter is: detected temperatures is 30 ℃, capillary column diameter 25-75 μ m, length 20mm, and pressure sample introduction, pressure are 0.5psi, sample injection time 5s-20s, WV 15-25kV.
Above-mentioned capillary column can use the coating quartz capillary column also can use the not quartz capillary column of coating.
Preferably, when using the coating quartz capillary column, above-mentioned electrophoresis WV is 20kV, and when using not the coating quartz capillary column, the electrophoresis WV is 25kV.
Preferably, electrophoretic was zone electrophoresis or gel electrophoresis when above-mentioned Capillary Electrophoresis detected, and its electrophoretic buffer is the inorganic salts damping fluid, and pH2.0-9.0 is preferred: phosphate, boric acid or citrate buffer solution.
Above-mentioned testing result is the collection of illustrative plates of sample separation, the utilization HPCE with analysis software, the peak height at each peak of integration or peak area with typical curve comparison, calculate sample concentration, take advantage of extension rate again, promptly get actual concentrations.
Detection method provided by the present invention is compared with existing method and is had the following advantages:
1. the sample centrifugation time is short, can not cause that Partial Protein deposition or fat are residual, and its centrifugal separating effect is better;
2. sample buffer can effectively prevent protein adsorption on capillary wall, can accurately reflect the protein actual content; Can make simultaneously various albumen keep separate state, remove phase mutual interference and gathering, compared with prior art, obviously improve the Separation of Proteins effect.In particular: compared with prior art, the raising of its urea content has improved absorbance, good separating effect, and separating absorbance has increased about 50%; Beta-mercaptoethanol obviously improves separating effect as the charged surface activating agent; It is better than using methyl hydroxyethylcellulose to use methyl hydroxyethylcellulose to form its separating effect of agent as dynamic network.
3. method of the present invention can use the coating quartz capillary column also can use the not quartz capillary column of coating, uses that the coating quartz capillary column is cheap, tough and tensile not durable, and environment for use is required the end, can reduce the detection cost.
4. method of the present invention can be used short quartz capillary column, and its velocity of separation is very fast.
5. method equation of linear regression related coefficient of the present invention>0.98; Its range of linearity is at 0.01mg/ml-2mg/ml, and minimum detectability is 0.15ng, and concentration limit is 0.005mg/ml; Degree of accuracy reaches more than 90%; The standard model recovery reaches more than 90%, and sample recovery rate reaches more than 70%, can effectively detect the content of the alpha-casein in the sample.
Description of drawings
Fig. 1 is the alpha-casein typical curve in the one embodiment of the invention;
Fig. 2 is that the alpha-casein in the one embodiment of the invention detects separating spectrum;
Fig. 3 is the alpha-casein standard typical curve in the another embodiment of the present invention;
Fig. 4 is that the alpha-casein in the another embodiment of the present invention detects separating spectrum;
Fig. 5 is that the alpha-casein among the another embodiment of the present invention detects separating spectrum;
Fig. 6 is that the alpha-casein among the another embodiment of the present invention detects separating spectrum;
Fig. 7 is that casein detects separating spectrum under the different centrifugal rotational speeds;
Fig. 8 is that casein detects separating spectrum under the different urea concentrations;
Fig. 9 uses beta-mercaptoethanol and DL-dithiothreitol (DTT) to detect separating spectrum as the casein of charged surface activating agent respectively;
Figure 10 is to use the casein of hydroxypropyl methylcellulose and methyl hydroxyethylcellulose to detect separating spectrum.
Embodiment
To combine specific embodiments that method of the present invention is done more detailed explanation below.It will be appreciated by those skilled in the art that following embodiment all is used for the present invention's scope required for protection is carried out the description of exemplary, summarize the relative scope of each parameter of the present invention, thereby can not it be interpreted as a kind of concrete restriction of the present invention with this.
Embodiment 1 detects alpha-casein content in the cow's milk (using the coating quartz capillary column)
1. the preparation of sample buffer: add 40mmol/L3-morpholino propane sulfonic acid in the 160mmol/L trimethyl aminomethane buffer solution; The 60mmol/L disodium ethylene diamine tetraacetate; 7mol/L urea; Add the beta-mercaptoethanol of 5ul/ml when handling sample, methyl hydroxyethylcellulose 0.05% uses the 0.1M sodium hydroxide solution to transfer to 8.5 to the pH value.
2. the preparation of electrophoretic buffer: 0.32mol/L citric acid, 20mmol/L trisodium citrate, 7mol/L urea, hydroxypropyl methylcellulose 0.05%.
3. high voltage capillary electrophoresis appearance (the P/ACE MDQ of U.S. Beckman Coulter Inc. HPCE); Detected parameters: detected temperatures is controlled at 30 ℃, and using diameter is 50 μ m, the length coating quartz capillary column as 20mm, PDAD (DAD detecting device); The pressure sample introduction; Sample introduction pressure is 0.5psi, and sample injection time is 5s, and WV is 20kV.
4. the formulation of typical curve: the alpha-casein standard items are dissolved in the sample buffer, are mixed with the standard solution that concentration is respectively 4mg/ml, 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml.The use eddy mixer shakes up, and the standard solution that accurately extracts after 40ul handles adds in the sample hose, uses aforementioned high voltage capillary electrophoresis appearance to measure afterwards.Utilize HPCE to carry software 32Karat
TM8.0 the peak area behind each the peak integration of collection of illustrative plates that obtains is respectively 2851350,1398500,740061,368065,150017, utilizes the high voltage capillary electrophoresis appearance to carry 32Karat
TMAs shown in Figure 1 (horizontal ordinate among the figure is represented sampled value, and unit is AU 8.0 software draws typical curve; Ordinate is represented match value, and unit/ml/L), its linear regression related coefficient is 0.9995, and minimum detectability is 0.01ug, and the minimum content that detects is 0.25mg/ml, and the standard model recovery reaches 91%.Before the separation, join electrophoretic buffer in the peculiar volumetric flask of instrument, to be detected.
5. sample preparation and detection: get in the centrifuge tube of milk sample adding 15ml centrifugal 10min under the 4500r/min centrifugal condition.Accurately extract centrifugal treating milk sample supernatant 100ul afterwards and insert frozen pipe (or other containers).Extract the 900ul sample buffer and add frozen pipe (or other containers), and use the eddy mixer mixing.The sample that accurately extracts after 40ul handles adds in the sample hose, is positioned over supersonic wave cleaning machine 3min, uses the high voltage capillary electrophoresis appearance to measure afterwards.
6. the quantitative reading of sample: obtain the collection of illustrative plates of sample separation after the instrument detecting, as shown in Figure 2 (horizontal ordinate among the figure is represented disengaging time, and unit is min; Ordinate is represented light absorption value, the AU of unit), the 32Karat that utilizes the high voltage capillary electrophoresis appearance to be carried
TM8.0 software, the peak area that obtains alpha-casein is 1036081, utilizes HPCE to carry 32Karat
TM8.0 software is inserted in automatically and calculates sample solution concentration in the above-mentioned typical curve equation shown in Figure 1 is 1.4mg/ml, takes advantage of extension rate 10 again, promptly getting actual concentrations is 14mg/ml, so the alpha-casein concentration that this milk appearance is contained is 14g/L.
Embodiment 2 detects alpha-casein content (using not coating quartz capillary column) in the cow's milk
1. the preparation of sample buffer: add 40mmol/L3-morpholino propane sulfonic acid in the 160mmol/L trimethyl aminomethane buffer solution; The 60mmol/L disodium ethylene diamine tetraacetate; 7mol/L urea, the beta-mercaptoethanol of interpolation 5ul/ml when handling sample, methyl hydroxyethylcellulose 0.05%.Using 0.1M NaOH to transfer to the pH value is 8.5.
2. the preparation of electrophoretic buffer: 0.32mol/L citric acid, 20mmol/L trisodium citrate, 7mol/L urea, hydroxypropyl methylcellulose 0.05%.
3. HPCE (the P/ACE MDQ of U.S. Beckman Coulter Inc. HPCE); Detected parameters: detected temperatures is controlled at 30 ℃, and using diameter is 75 μ m, the length not coating quartz capillary column as 600mm, the DAD detecting device; The pressure sample introduction; Sample introduction pressure is 0.5psi, and sample injection time is 5s, and WV is 25kV.
4. the formulation of calibration curve: the alpha-casein standard items are dissolved in the sample buffer, are mixed with the standard solution that concentration is respectively 4mg/ml, 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml.The use eddy mixer shakes up, and the standard solution that accurately extracts after 40ul handles adds in the sample hose, uses the high voltage capillary electrophoresis appearance to measure.Peak area behind each peak integration of the collection of illustrative plates that detection obtains is respectively, and 2951350,1487620,751200,369034,140010, utilize 32Karat
TM8.0 it is as shown in Figure 3 that analysis software draws typical curve, its linear regression related coefficient is 0.9996, and the minimum line that detects is 0.01ug, and the minimum content that detects is 0.25mg/ml, and the standard model recovery reaches 90%.Before the separation, join electrophoretic buffer in the peculiar volumetric flask of instrument, to be detected.Horizontal ordinate among the figure is represented sampled value, and unit is AU; Ordinate is represented match value, unit/ml/L.
5. sample preparation and detection: get in the centrifuge tube of milk sample adding 15ml centrifugal 5min under the 4500r/min centrifugal condition.Accurately extract centrifugal treating milk sample supernatant 100ul afterwards and insert frozen pipe (or other containers).Extract the 900ul sample buffer and add frozen pipe (or other containers), and use the eddy mixer mixing.Accurately extract 40ul and handle in the sample adding sample hose, be positioned over supersonic wave cleaning machine 3min, use HPCE to measure afterwards.
6. the quantitative reading of sample: the collection of illustrative plates that obtains sample separation after the instrument detecting; As shown in Figure 4, the utilization HPCE with analysis software, the peak area of integration alpha-casein is 1124510; Utilize analysis software to be inserted in automatically to calculate sample solution concentration in the above-mentioned typical curve equation shown in Figure 3 and be 1.49mg/ml; Take advantage of extension rate 10 again, promptly getting actual concentrations is 14.9mg/ml, so the alpha-casein concentration that this milk appearance is contained is 14.9g/L.
The detection method of 3 one kinds of pasteurization Ruzhongs of embodiment alpha-casein content
Use sample buffer, electrophoretic buffer and the high voltage capillary electrophoresis appearance identical, the instrument detecting parameter that use and embodiment 1 are identical with embodiment 1.The typical curve that adopts embodiment 1 to obtain.
Sample preparation with the detection step is: add in the centrifuge tube of 15ml centrifugal 10min under the 4500r/min centrifugal condition to pasteurization breast sample.Accurately extract centrifugal treating pasteurization breast sample supernatant 100ul afterwards and insert frozen pipe (or other containers).Extract the 300ul sample buffer and add frozen pipe (or other containers), and use the eddy mixer mixing.Accurately extract 40ul and handle in the sample adding sample hose, be positioned over supersonic wave cleaning machine 3min, use HPCE to measure afterwards.
The quantitative reading of sample: the collection of illustrative plates that obtains sample separation after the instrument detecting; As shown in Figure 5, the utilization HPCE with analysis software, the peak area of integration alpha-casein is 192993; Utilize analysis software to be inserted in automatically to calculate sample solution concentration in the typical curve equation shown in Figure 1 and be 2.76mg/ml; Take advantage of extension rate 5 again, promptly getting actual concentrations is 13.8mg/ml, so the alpha-casein concentration that this sample contains is 13.8g/L.
The detection method of alpha-casein content in 4 one kinds of UHT sterile milks of embodiment
Use sample buffer, electrophoretic buffer and the high voltage capillary electrophoresis appearance identical, use the instrument detecting parameter identical with embodiment with embodiment 1.The typical curve that adopts embodiment 1 to obtain.
Sample preparation with the detection step is: add UHT sterile milk sample in the centrifuge tube of 15ml centrifugal 10min under the 4500r/min centrifugal condition.Accurately extract centrifugal treating UHT sterile milk sample supernatant 50ul afterwards and insert frozen pipe (or other containers).Extract the 950ul sample buffer and add frozen pipe (or other containers), and use the eddy mixer mixing.Accurately extract 40ul and handle in the sample adding sample hose, be positioned over supersonic wave cleaning machine 3min, use HPCE to measure afterwards.
The quantitative reading of sample: the collection of illustrative plates that obtains sample separation after the instrument detecting; As shown in Figure 6, the utilization HPCE with analysis software, the peak area of integration alpha-casein is 574181; Utilize analysis software to be inserted in automatically to calculate sample solution concentration in the typical curve equation shown in Figure 1 and be 0.78mg/ml; Take advantage of extension rate 20 again, promptly getting actual concentrations is 15.6mg/ml, so the alpha-casein concentration that this sample contains is 15.6g/L.
The detection method of alpha-casein content in 5 one kinds of raw milk of embodiment
The method of sample buffer, electrophoretic buffer and specified value curve is identical with embodiment 1.High voltage capillary electrophoresis appearance (the P/ACE MDQ of U.S. Beckman Coulter Inc. HPCE); Detected parameters: detected temperatures is controlled at 15 ℃, and using diameter is 25 μ m, the length not coating quartz capillary column as 60mm, UV-detector; Electrokinetic injection; Sample introduction pressure is 0psi, and sample injection time is 20s, and WV is 7kV.
Detection obtains typical curve, and its linear regression related coefficient is 0.9996, and minimum detectability is 0.01ug, and the minimum content that detects is 0.15mg/ml, and the standard model recovery reaches 90%, sample recovery rate 70%.
Sample preparation and detection: get in the centrifuge tube of raw milk sample adding 15ml centrifugal 15min under the 2000r/min centrifugal condition.Accurately extract centrifugal treating raw milk sample supernatant 40ul afterwards and insert frozen pipe (or other containers).Extract the 800ul sample buffer and add frozen pipe (or other containers), and use the eddy mixer mixing.Accurately extract 40ul and handle in the sample adding sample hose, be positioned over supersonic wave cleaning machine 1min, use HPCE to measure afterwards.
The quantitative reading of sample: the collection of illustrative plates that obtains sample separation after the instrument detecting; The utilization HPCE with analysis software; The peak area of integration alpha-casein is 1134560, utilizes analysis software to be inserted in automatically to calculate sample solution concentration in the typical curve equation to be 1.69mg/ml, takes advantage of extension rate 21 again; Promptly getting actual concentrations is 35.49mg/ml, so the alpha-casein concentration that this milk appearance is contained is 35.49g/L.
The detection method of alpha-casein content in 6 one kinds of raw milk of embodiment
The method of sample buffer, electrophoretic buffer and specified value curve is identical with embodiment 1.High voltage capillary electrophoresis appearance (the P/ACE MDQ of U.S. Beckman Coulter Inc. HPCE); Detected parameters: detected temperatures is controlled at 15 ℃, and using diameter is 50 μ m, the length not coating quartz capillary column as 60mm, UV-detector; The pressure sample introduction; Sample introduction pressure is 1psi, and sample injection time is 1s, and WV is 30kV.
Detection obtains typical curve, and its linear regression related coefficient is 0.9991, and minimum detectability is 0.01ug, and the minimum content that detects is 0.15mg/ml, and the standard model recovery reaches 92%, sample recovery rate 90%.
Sample preparation and detection: get in the centrifuge tube of raw milk sample adding 15ml centrifugal 5min under the 6000r/min centrifugal condition.Accurately extract centrifugal treating raw milk sample supernatant 100ul afterwards and insert frozen pipe (or other containers).Extract the 900ul sample buffer and add frozen pipe (or other containers), and use the eddy mixer mixing.Accurately extract 40ul and handle in the sample adding sample hose, be positioned over supersonic wave cleaning machine 10min, use HPCE to measure afterwards.
The quantitative reading of sample: the collection of illustrative plates that obtains sample separation after the instrument detecting; The utilization HPCE with analysis software; The peak area of integration alpha-casein is 11563020, utilizes analysis software to be inserted in automatically to calculate sample solution concentration in the typical curve equation to be 1.32mg/ml, takes advantage of extension rate 10 again; Promptly getting actual concentrations is 13.20mg/ml, so the alpha-casein concentration that this milk appearance is contained is 13.20g/L.
Embodiment 7 comparative examples
1. separating effect contrast under the different centrifugal rotational speeds:
Method according to embodiment 1 makes an experiment, the adjustment centrifugal rotational speed, and the result is as shown in Figure 7: the alpha-casein good separating effect is followed successively by 4500r/min, 4000r/min and 3500r/min to the order of difference.
2. when forming, separate different sample buffers the contrast of effect:
1) under the different urea contents, the separating effect contrast, concrete experimental procedure is: fixing other compositions; Use urea concentration to handle sample respectively as the sample buffer of 7mol/L and 6mol/L; Detect former milk, it is as shown in Figure 8 to obtain alpha-casein, and urea content is that the light absorption value of 7mol/L is higher than the light absorption value of 6mol/L; And whole collection of illustrative plates is clear, separating effect is better, and separating light absorption value increases about 50%.
2) separating effect of different electric charge surfactants contrast, fixing other compositions use beta-mercaptoethanol and DL-dithiothreitol (DTT) (DTT) respectively, more than two kinds of materials be the charged surface activating agent.But the beta-mercaptoethanol effect is bigger six times than DTT, and the stronger separating effect of function is good more obviously.It is as shown in Figure 9 to detect the collection of illustrative plates that obtains after the former milk, and the visible collection of illustrative plates that uses beta-mercaptoethanol is obviously than the good separating effect that uses DTT.
3) use the separating effect of hydroxypropyl methylcellulose and methyl hydroxyethylcellulose to contrast, both are dynamic network and form agent.Separate pasteurization milk, the separating spectrum that obtains is shown in figure 10: can see, use the hydroxypropyl methylcellulose ratio to use the methyl hydroxyethylcellulose separating effect better.
Claims (10)
1. the detection method of a Ruzhong alpha-casein content; May further comprise the steps: the newborn sample and the sample buffer that will detect carry out hybrid processing; Sample after the hybrid processing is detected through HPCE; It is characterized in that the compound method of said sample buffer is: 160mmol/L trimethyl aminomethane buffer solution adds 40mmol/L3-morpholino propane sulfonic acid, 60mmol/L disodium ethylene diamine tetraacetate, 7mol/L urea, adds the beta-mercaptoethanol of 5ul/ml when handling sample again; Methyl hydroxyethylcellulose 0.05% is regulated pH value to 8.5; The detected parameters of said HPCE is: detected temperatures 15-40 ℃; Capillary column diameter 25-75 μ m, length 20-600mm, PDAD or UV-detector, pressure sample introduction or electrokinetic injection; Pressure is 0-1psi; Sample injection time 1s-20s, detected temperatures is 15-40 ℃, WV 7-30kV.
2. detection method according to claim 1 is characterized in that, before said newborn sample and said sample buffer carry out hybrid processing, said newborn sample is carried out centrifugal treating, and centrifugal condition is: rotating speed 2000-6000r/min, centrifugal 5-15min.
3. detection method according to claim 2, wherein, said centrifugal condition is: rotating speed 4500r/min, centrifugation time 10min.
4. detection method according to claim 2 is characterized in that, before the sample after the said hybrid processing being detected through said HPCE, with the sample after the said hybrid processing with ultrasonic cleaning 1-10min.
5. according to the described detection method of one of claim 1-4; It is characterized in that; Carry out in the step of hybrid processing at said newborn sample and said sample buffer; Get said newborn sample or centrifugal after supernatant and said sample buffer carry out hybrid processing, the supernatant of newborn sample and the blending ratio of said sample buffer are 1: (3-20) (v/v).
6. detection method according to claim 5; It is characterized in that; This method further comprises the steps: 1) alpha-casein is dissolved in the said sample buffer; Prepare the standard solution of a plurality of different content alpha-caseins, and said a plurality of standard solution are detected the typical curve that obtains between alpha-casein content and the testing result respectively through said HPCE; 2) will detect said newborn test result of samples and the comparison of said typical curve that obtains through said HPCE, obtain the content of alpha-casein in the said newborn sample.
7. detection method according to claim 6 is characterized in that, in said step 1), in said sample buffer, dissolves the alpha-casein standard substance, is mixed with the standard solution of 4mg/ml, 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml.
8. detection method according to claim 7 is characterized in that, the supernatant of said newborn sample or said alpha-casein standard substance and said sample buffer use eddy mixer to shake up.
9. detection method according to claim 1, wherein, the detected parameters of said HPCE is: 30 ℃ of detected temperatures; Capillary column diameter 25-75 μ m, length 20mm, pressure sample introduction, pressure are 0.5psi; Sample injection time 5s-20s, WV 15-25kV.
10. detection method according to claim 1 is characterized in that, said capillary column is the coating quartz capillary column, and said WV is 20kV; Or said capillary column is coating quartz capillary column not, and said WV is 25kV.
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| CN101793866A (en) * | 2010-03-29 | 2010-08-04 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting content of immunoglobulin IgG in human milk |
| CN101806770A (en) * | 2010-03-29 | 2010-08-18 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting immunoglobulin IgA content in human milk |
| CN105136896B (en) * | 2015-08-19 | 2017-11-10 | 内蒙古蒙牛乳业（集团）股份有限公司 | The method for determining κ casein contents in cow's milk |
| CN106596818B (en) * | 2015-12-17 | 2018-05-22 | 中国医科大学 | The kit and assay method of the identification of chicken serum albumin and absolute quantitation can be achieved |
| CN110346443B (en) * | 2018-04-04 | 2021-11-09 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting A2 beta-casein content in cow milk |
| CN117169317B (en) * | 2023-10-30 | 2024-01-19 | 内蒙古蒙牛乳业（集团）股份有限公司 | Alpha-s 1 casein phosphorylation degree detection method, sample buffer solution and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1412558A (en) * | 2001-10-15 | 2003-04-23 | 中国农业大学 | Macromolecular weight protein reference material for active electrophoresis |
| CN1608202A (en) * | 2001-12-28 | 2005-04-20 | 独立行政法人科学技术振兴机构 | Method of electrophoresing protein |
| US20050148021A1 (en) * | 2001-12-05 | 2005-07-07 | Pier Righetti G. | Process for the selective alkylation of-sh groups in proteins and peptides for the study of complex protein mixtures |
| US20050274616A1 (en) * | 2004-05-10 | 2005-12-15 | Sebia | Method for separating proteins by capillary electrophoresis and buffer compositions for capillary electrophoresis |
-
2008
- 2008-08-28 CN CN2008101469514A patent/CN101339159B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1412558A (en) * | 2001-10-15 | 2003-04-23 | 中国农业大学 | Macromolecular weight protein reference material for active electrophoresis |
| US20050148021A1 (en) * | 2001-12-05 | 2005-07-07 | Pier Righetti G. | Process for the selective alkylation of-sh groups in proteins and peptides for the study of complex protein mixtures |
| CN1608202A (en) * | 2001-12-28 | 2005-04-20 | 独立行政法人科学技术振兴机构 | Method of electrophoresing protein |
| US20050274616A1 (en) * | 2004-05-10 | 2005-12-15 | Sebia | Method for separating proteins by capillary electrophoresis and buffer compositions for capillary electrophoresis |
Non-Patent Citations (1)
| Title |
|---|
| 张东送 等.毛细管电泳在牛乳中酪蛋白含量测定及掺假检测方面的应用.《食品与发酵工业》.中国食品发酵工业研究院，全国食品与发酵工业信息中心,2005,第31卷(第1期),130-132. * |
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