CN101339159A - 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 uses widely in food industry and other industrial all having obtained.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, and in conjunction with 8 phosphate anions, binding site mainly concentrates on the 43-79 position of peptide chain on each molecule, 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 ".
Capillary electrophoresis apparatus is that a class comprises that the liquid phase differential of 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.Capillary electrophoresis apparatus 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 only is about 1-2h.At present, the application prospect of capillary electrophoresis apparatus 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 precipitation 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 problem to be solved in the present invention 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 by 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 need to detect carry out hybrid processing, sample after the hybrid processing is detected by capillary electrophoresis apparatus, the compound method of described sample buffer is: the trimethyl aminomethane buffer solution adds 3-morpholino propane sulfonic acid, disodium ethylene diamine tetraacetate, urea, add beta-mercaptoethanol, methyl hydroxyethylcellulose when handling sample again.
Preferably, the compound method of sample buffer is: 160mmol/L trimethyl aminomethane buffer solution adds 40mmol/L 3-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 complete 8.5.
Preferably, with before described sample buffer mixes, newborn sample is carried out centrifugal treating at described 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 described hybrid processing being detected by described capillary electrophoresis apparatus, with the sample after the described 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, described detection method further comprises the steps: 1) alpha-casein is dissolved in the described sample buffer, prepare the standard solution of a plurality of different content alpha-caseins, and described a plurality of standard solution are detected the typical curve that obtains between alpha-casein content and the testing result respectively by described capillary electrophoresis apparatus; 2) will detect described newborn test result of samples and the comparison of described typical curve that obtains by described capillary electrophoresis apparatus, obtain the content of alpha-casein in the described newborn sample.
Above-mentioned steps 1) in, in described 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 described newborn sample or described alpha-casein standard substance and described sample buffer use eddy mixer to shake up.This instrument energy biased sample is more even, can not produce any destruction to sample simultaneously.
Above-mentioned capillary electrophoresis apparatus detected parameters is as follows: detected temperatures 15-40 ℃, capillary column diameter 25-75 μ m, length 20-600mm, diode array detector or UV-detector, pressure sample introduction or electrokinetic injection, pressure is 0-1psi, sample injection time 1s-20s, detected temperatures is 15-40 ℃, operating voltage 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, operating voltage 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 operating voltage is 20kV, and when using not the coating quartz capillary column, the electrophoresis operating voltage 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 capillary electrophoresis apparatus 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 precipitation 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, minimum detectability is 0.15ng, 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 in the another embodiment of the present invention detects separating spectrum;
Fig. 6 is that the alpha-casein in 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
Below in conjunction with specific embodiments method of the present invention is done more detailed description.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 it can not be interpreted as a kind of concrete restriction of the present invention with this.
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 that the pH value is transferred to 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. high voltage capillary electrophoresis instrument (the P/ACE MDQ of U.S. Beckman Coulter Inc. capillary electrophoresis apparatus), detected parameters: detected temperatures is controlled at 30 ℃, using diameter is 50 μ m, the length coating quartz capillary column as 20mm, diode array detector (DAD detecting device), the pressure sample introduction, sample introduction pressure is 0.5psi, and sample injection time is 5s, and operating voltage 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 instrument to measure afterwards.Utilize capillary electrophoresis apparatus 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 instrument to carry 32Karat
TM(horizontal ordinate among the figure is represented sampled value, and unit is AU as shown in Figure 1 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, electrophoretic buffer is joined 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 instrument to measure afterwards.
6. the quantitative reading of sample: obtain the collection of illustrative plates of sample separation after the instrument detecting, (horizontal ordinate among the figure is represented disengaging time, and unit is min as shown in Figure 2; Ordinate is represented light absorption value, the AU of unit), the 32Karat that utilizes the high voltage capillary electrophoresis instrument to be carried
TM8.0 software, the peak area that obtains alpha-casein is 1036081, utilizes capillary electrophoresis apparatus 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 sample contains is 14g/L.
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. capillary electrophoresis apparatus (the P/ACE MDQ of U.S. Beckman Coulter Inc. capillary electrophoresis apparatus), detected parameters: detected temperatures is controlled at 30 ℃, 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 operating voltage 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 instrument 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 analysis software draws typical curve as shown in Figure 3, 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, electrophoretic buffer is joined 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 capillary electrophoresis apparatus 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 capillary electrophoresis apparatus 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 sample contains 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 instrument 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 and detection step are: pasteurization breast sample is added in the centrifuge tube of 15ml centrifugal 10min under the 4500r/min centrifugal condition.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 capillary electrophoresis apparatus 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 capillary electrophoresis apparatus 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 instrument identical, use the instrument detecting parameter identical with embodiment with embodiment 1.The typical curve that adopts embodiment 1 to obtain.
Sample preparation and detection step are: UHT sterile milk sample is added 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 capillary electrophoresis apparatus 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 capillary electrophoresis apparatus 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 instrument (the P/ACE MDQ of U.S. Beckman Coulter Inc. capillary electrophoresis apparatus), detected parameters: detected temperatures is controlled at 15 ℃, 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 operating voltage 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 capillary electrophoresis apparatus to measure afterwards.
The quantitative reading of sample: the collection of illustrative plates that obtains sample separation after the instrument detecting, the utilization capillary electrophoresis apparatus with analysis software, the peak area of integration alpha-casein is 1134560, utilize analysis software to be inserted in automatically to calculate sample solution concentration in the typical curve equation and be 1.69mg/ml, take advantage of extension rate 21 again, promptly getting actual concentrations is 35.49mg/ml, so the alpha-casein concentration that this milk sample contains 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 instrument (the P/ACE MDQ of U.S. Beckman Coulter Inc. capillary electrophoresis apparatus), detected parameters: detected temperatures is controlled at 15 ℃, 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 operating voltage 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 capillary electrophoresis apparatus to measure afterwards.
The quantitative reading of sample: the collection of illustrative plates that obtains sample separation after the instrument detecting, the utilization capillary electrophoresis apparatus with analysis software, the peak area of integration alpha-casein is 11563020, utilize analysis software to be inserted in automatically to calculate sample solution concentration in the typical curve equation and be 1.32mg/ml, take advantage of extension rate 10 again, promptly getting actual concentrations is 13.20mg/ml, so the alpha-casein concentration that this milk sample contains is 13.20g/L.
Embodiment 7 comparative examples
1. separating effect contrast under the different centrifugal rotational speeds:
Method according to embodiment 1 is tested, and adjusts centrifugal rotational speed, the result 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 that differs from.
2. when forming, separate different sample buffers the contrast of effect:
1) under the different urea content, the separating effect contrast, concrete experimental procedure is: fix other compositions, use urea concentration to handle sample respectively as the sample buffer of 7mol/L and 6mol/L, detect former milk, obtain alpha-casein as shown in Figure 8, urea content is the light absorption value height of the light absorption value of 7mol/L than 6mol/L, and whole collection of illustrative plates is clear, separating effect is better, and separating light absorption value increases about 50%.
2) other compositions are fixed in the separating effect of different charged surface activating agents contrast, 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 strong more separating effect of function is good more obviously.Detect the collection of illustrative plates that obtains after the former milk as shown in Figure 9, the collection of illustrative plates that as seen 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 as 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 need to detect carry out hybrid processing, sample after the hybrid processing is detected by capillary electrophoresis apparatus, it is characterized in that, the compound method of described sample buffer is: the trimethyl aminomethane buffer solution adds 3-morpholino propane sulfonic acid, disodium ethylene diamine tetraacetate, urea, add beta-mercaptoethanol, methyl hydroxyethylcellulose when handling sample again.
2. detection method according to claim 1, it is characterized in that, the compound method of described 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.
3. detection method according to claim 1 and 2 is characterized in that, before described newborn sample and described sample buffer carry out hybrid processing, described newborn sample is carried out centrifugal treating, and centrifugal condition is: rotating speed 2000-6000r/min, centrifugal 5-15min; Preferred rotating speed 4500r/min, centrifugation time 10min.
4. detection method according to claim 3 is characterized in that, before the sample after the described hybrid processing being detected by described capillary electrophoresis apparatus, with the sample after the described 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 described newborn sample and described sample buffer, get described newborn sample or centrifugal after supernatant and described sample buffer carry out hybrid processing, the supernatant of newborn sample and the blending ratio of described 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 described sample buffer, prepare the standard solution of a plurality of different content alpha-caseins, and described a plurality of standard solution are detected the typical curve that obtains between alpha-casein content and the testing result respectively by described capillary electrophoresis apparatus; 2) will detect described newborn test result of samples and the comparison of described typical curve that obtains by described capillary electrophoresis apparatus, obtain the content of alpha-casein in the described newborn sample.
7. detection method according to claim 6 is characterized in that, in described step 1), dissolves the alpha-casein standard substance in described sample buffer, 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 described newborn sample or described alpha-casein standard substance and described sample buffer use eddy mixer to shake up.
9. according to claim 7 or 8 described detection methods, it is characterized in that, the detected parameters of described capillary electrophoresis apparatus is: detected temperatures 15-40 ℃, capillary column diameter 25-75 μ m, length 20-600mm, diode array detector or UV-detector, pressure sample introduction or electrokinetic injection, pressure is 0-1psi, sample injection time 1s-20s, detected temperatures is 15-40 ℃, operating voltage 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, operating voltage 15-25kV.
10. detection method according to claim 9 is characterized in that, described capillary column is the coating quartz capillary column, and described operating voltage is 20kV; Or described capillary column is coating quartz capillary column not, and described operating voltage is 25kV.
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| 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 |
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| 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 |
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| CN1159579C (en) * | 2001-10-15 | 2004-07-28 | 中国农业大学 | Macromolecular weight protein reference material for active electrophoresis |
| ITMI20012564A1 (en) * | 2001-12-05 | 2003-06-05 | Pier Giorgio Righetti | PROCEDURE FOR THE SELECTIVE ALKYLATION OF SH-GROUPS IN PROTEINS AND PEPTIDES IN THE STUDY OF COMPLEX PROTEIN MIXTURES |
| JP2003194775A (en) * | 2001-12-28 | 2003-07-09 | Japan Science & Technology Corp | Electrophoresis method of protein |
| FR2869995B1 (en) * | 2004-05-10 | 2006-09-22 | Sebia Sa | IMPROVED CAPILLARY ELECTROPHORESIS PROTEIN SEPARATION PROCESS AND CAPILLARY ELECTROPHORESIS BUFFER COMPOSITIONS |
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| CN117169317A (en) * | 2023-10-30 | 2023-12-05 | 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 |
| 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 |
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