CN101294931B - Method for detecting content of beta-lactoglobulin in cow's milk - Google Patents
Method for detecting content of beta-lactoglobulin in cow's milk Download PDFInfo
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Abstract
The invention relates to a method for detecting components in milk, in particular to a method for detecting the content of Beta-lactoglobulin, belonging to the detection technical field; the detection method using a capillary electrophoresis meter to detect the content of Beta-lactoglobulin of the invention comprises the following steps: the pretreatment of samples, detection of sample-loading, alignment of reading and edit of report, etc.; the invention is that the samples are processed by electrophoresis on the capillary electrophoresis meter, thus detecting absorbance of the samples by a capillary chromatographic system, expressing the absorbance by the mode of mapping, detecting the content of Beta-lactoglobulin accurately and providing the technical support for the development of products, quality control and analysis of the components.
Description
Technical field
The present invention relates to the method for a kind of analyzing and testing Ruzhong albumen, particularly a kind of method that detects beta-lactoglobulin content in the cow's milk.Belong to the detection technique field.
Background technology
Beta lactoglobulin is a kind of important milk albumin, accounts for 8%～10% of gross protein massfraction, 20-40mg/ml.The beta lactoglobulin molecular weight is about 18400D, mainly is made up of 162 amino acid.The secondary structure of beta lactoglobulin is made up of the molecule of 9 antiparallel beta sheets and 1 one spiral.Beta lactoglobulin is the main sensibiligen of baby's milk allergy.This is because beta lactoglobulin is attracted on the intestinal mucosa, produces immune response and causes allergy.Help to improve baby's allergy so reduce the content of beta lactoglobulin.Therefore accurately detecting beta-lactoglobulin content has very important meaning.
National standard and industry standard that the beta-lactoglobulin content detection method is not arranged at present, as yet.Domestic relevant criterion has the mensuration of GB/T 5413.1,2-1997 dispensed food for baby and milk powder-lactalbumin.The detection method of beta lactoglobulin commonly used is spectrophotometer method, ELISA and high performance liquid chromatography.It is highly sensitive that immuno-enzymatic joins absorption method, and minimum detectable level is low, is suitable for the whole process analysis of beta lactoglobulin separation and purification; High performance liquid chromatography quick and precisely, and is easy and simple to handle, the Synchronization Analysis in the time of can realizing the beta lactoglobulin initial gross separation; AAS is fast and convenient, and accuracy is poor slightly, is suitable for having behind the purifying on-line analysis of the beta lactoglobulin of higher concentration.
Summary of the invention
The object of the invention: the detection method of beta-lactoglobulin content in a kind of cow's milk.Relative merits in view of method in the prior art; This patent has been invented the method that will use HPCE fast detecting breast beta-lactoglobulin content; And a series of operation parameter and dedicated buffering liquid have creatively been tested to this instrument; Making that the accuracy and the speed that detect are all very desirable, is creative place of the present invention.
The present invention is a kind of detection method that detects beta-lactoglobulin content; Wherein, Comprise the steps: 1) beta lactoglobulin is dissolved in sample buffer; Prepare the standard solution of a plurality of different content beta lactoglobulins, and obtain the typical curve between beta-lactoglobulin content and the testing result after a plurality of solution are detected respectively; 2) sample is detected, and obtain testing result; 3), obtain the content of beta lactoglobulin in the sample with described testing result and the comparison of described typical curve; Wherein, the equipment of test sample is the high voltage capillary electrophoresis analyser.
Above-mentioned steps 1) method of the formulation of typical curve is preferably following in: preparation beta lactoglobulin standard solution; Dissolving beta lactoglobulin standard substance is mixed with and is respectively 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml in sample buffer; Prepare standard solution; Shake up the back and use the high voltage capillary electrophoresis analysis, obtain testing result, promptly obtain the relation curve of the beta-lactoglobulin content and the average correction factor; Wherein, described testing result is calculated according to following method: go out the average correction factor by calculated by peak area.
The above-mentioned standard solution or the disposal route of detected sample are: pack standard solution or detected sample in the centrifuge tube into; Centrifugal 5-15min under the 2000-6000r/min centrifugal condition; With sample and damping fluid according to 1: the ratio of 4-20 adds frozen pipe (or other containers), and shakes up, and accurately extracts 10-200ul and handles sample and add in the sample hose; Be positioned over supersonic wave cleaning machine 1～10min, use HPCE to measure.
Above-mentioned sample buffer is: the damping fluid that protein is disperseed, and preferred following damping fluid:
Add 40mmol/L 3-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%, pH value are 8.5
The above-mentioned step that shakes up adopts eddy mixer.
The use detected parameters that above-mentioned high voltage capillary electrophoresis is analyzed is following: the temperature that Capillary Electrophoresis detects is controlled between 15～40 ℃; Capillary column diameter 25～75 μ m, length 20～600mm, DAD (or ultraviolet light) detecting device, pressure sample introduction (or electrokinetic injection); Pressure is 0～1psi; Sample injection time is 1s～20s, and separation temperature is 15～40 ℃, and WV is 10～30kV.
Above-mentioned capillary column is a quartz capillary column, comprises coating and coating not.
Electrophoretic is a zone electrophoresis during detection of above-mentioned high voltage capillary electrophoresis analysis, and its electrophoretic buffer is pH2.0～9.0, and principal ingredient is the inorganic salts damping fluid, and is preferred: phosphate, boric acid or citric acid.
The disposal route of above-mentioned testing result is following: testing result is the collection of illustrative plates of sample separation, the utilization HPCE with analysis software, the area at each peak of integration calculates sample solution concentration, takes advantage of extension rate again, promptly gets actual concentrations.
Beta lactoglobulin is a kind of important milk albumin, accounts for 8%～10% of normal newborn gross protein massfraction, 20-40mg/ml.HPCE detects least concentration can reach 10
-5～10
-8Mol/L.So HPCE can detect its content.The method of using the HPCE exploitation to detect beta-lactoglobulin content comprise properties of samples understanding, clastotype, set up buffer system, optimize buffer system and change step such as clastotype.The method of developing can be applied in product development, quality control and the analyzing and testing field.
The present invention mainly uses HPCE can accurately measure the content of beta lactoglobulin, thereby is applied in product development, quality control and the analyzing and testing field.
The present invention uses the high voltage capillary electrophoresis appearance to detect the content of beta lactoglobulin on the basis in the principle of traditional electrophoresis and chromatogram, reaches the content that accurately detects beta lactoglobulin in the dairy products.Through test, finally confirm the optimal parameter condition of detection method.Specific as follows:
Detect the detection method of beta-lactoglobulin content, comprise sample pre-treatments, go up the appearance detection, also comprise steps such as calibration reading.The temperature that Capillary Electrophoresis detects is controlled between 15～40 ℃; The quartz capillary column of capillary column diameter 50～75 μ m, length 20～600mm, DAD (or ultraviolet light) detecting device, pressure sample introduction; Pressure is 0.5psi; Sample injection time is 5s～20s, and separation temperature is 20～40 ℃, and WV is 15-25kV.
The formulation of calibration curve: preparation beta lactoglobulin concentration of standard solution is respectively 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml, 0.051mg/ml.Shake up the back and use the high voltage capillary electrophoresis analysis, calculate the average correction factor by peak height.
The detection step is:
Pack the raw material milk sample in the centrifuge tube of 15ml into centrifugal 5min under the 5000r/min centrifugal condition.Accurately extract sample 100ul and insert frozen pipe.Extract the 400ul 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.
The quantitative reading of sample: the collection of illustrative plates that separates per sample, the utilization HPCE with analysis software, each peak height of integration (or peak area) calculates sample solution concentration, takes advantage of extension rate again, promptly gets actual concentrations.
Beneficial effect
The existing regression equation related coefficient of the inventive method＞0.98, its range of linearity are at 0.01mg/ml～2mg/ml, and the minimum line that detects is 0.15ng, and concentration limit is 0.005mg/ml.Degree of accuracy reaches more than 90%, and the pillar recovery reaches more than 90%, and sample recovery rate reaches more than 70%.Can effectively detect the content of the beta lactoglobulin in the sample.
This patent has been invented the method that will use HPCE fast detecting beta-lactoglobulin content.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.This method is easy and simple to handle, accurate, and detecting out as a result, the time is merely about 1h.
Embodiment
The detection method of beta-lactoglobulin content in 1 one kinds of cow's milk of embodiment
The Capillary Electrophoresis temperature is controlled at 25 ℃, and using diameter is 50 μ m, the length quartz capillary column as 20mm, and DAD detecting device, sample introduction pressure are 0.5psi, and sample injection time is 10s, and WV is 15kV.
The formulation of calibration curve: utilize beta lactoglobulin standard items compound concentration to be respectively the standard solution of 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml.Shake up the back and use the high voltage capillary electrophoresis analysis; Peak area behind the integration is respectively 378089,152088,72110,33124,14112; Utilize software to draw typical curve, the linear regression related coefficient is 0.9917, and the minimum line that detects is 0.01ug; The minimum content that detects is 0.01mg/ml, and the standard model recovery reaches 90%.
Handle the sample buffer collocation method: add 40mmol/L 3-morpholino propane sulfonic acid in the 160mmol/L trimethyl aminomethane buffer solution; The 60mmol/L disodium ethylene diamine tetraacetate; 6mol/L urea; Add the beta-mercaptoethanol of 5ul/ml when handling sample, methyl hydroxyethylcellulose 0.05% uses sodium hydroxide solution to transfer to 8.5 to the pH value.
The collocation method of electrophoretic buffer: 0.32mol/L citric acid, 20mmol/L trisodium citrate, 7mol/L urea, hydroxypropyl methylcellulose 0.05%.
The detection step is: add the milk sample in the centrifuge tube of 15ml centrifugal 10min under the 5000r/min centrifugal condition.Accurately extract sample 100ul and insert frozen pipe (or other containers).Extract the 400ul 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 separates per sample; The utilization HPCE with analysis software; The peak area of integration beta lactoglobulin is 478090, utilizes analysis software to be inserted in automatically to calculate sample solution concentration in the typical curve equation to be 2.7mg/ml, takes advantage of extension rate 5 again; Promptly getting actual concentrations is 13.5mg/ml, so the beta lactoglobulin concentration that this milk appearance is contained is 13.5g/L.
The detection method of beta-lactoglobulin content in 2 one kinds of cow's milk of embodiment
The Capillary Electrophoresis temperature is controlled at 30 ℃, and using diameter is 75 μ m, the length quartz capillary column as 600mm, and DAD detecting device, sample introduction pressure are 0.5psi, and sample injection time is 5s, and WV is 25kV.
The formulation of calibration curve: utilize beta lactoglobulin standard items compound concentration to be respectively the standard solution of 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml.Shake up the back and use the high voltage capillary electrophoresis analysis; Peak area behind the integration is respectively, and 393142,178563,87892,43728,21157, utilize software to draw typical curve; The linear regression related coefficient is 0.998; The minimum line that detects is 0.01ug, and the minimum content that detects is 0.01mg/ml, and the standard model recovery reaches 95%.
Handle the sample buffer collocation method: add 40mmol/L 3-morpholino propane sulfonic acid in the 160mmol/L trimethyl aminomethane buffer solution; The 60mmol/L ethylenediamine tetraacetic acid; 6mol/L urea, the beta-mercaptoethanol of interpolation 5ul/ml when handling sample, methyl hydroxyethylcellulose 0.4%.Using 0.1M NaOH to transfer to the pH value is 8.5.
The collocation method of electrophoretic buffer: 0.32mol/L citric acid, 20mmol/L trisodium citrate, 6mol/L urea, hydroxypropyl methylcellulose 0.4%.
The detection step is: add the milk sample in the centrifuge tube of 15ml centrifugal 5min under the 5000r/min centrifugal condition.Accurately extract sample 100ul and insert frozen pipe (or other containers).Extract the 400ul 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 separates per sample; The utilization HPCE with analysis software; The peak area of integration beta lactoglobulin is 514202, utilizes analysis software to be inserted in automatically to calculate sample solution concentration in the typical curve equation to be 2.6mg/ml, takes advantage of extension rate 5 again; Promptly getting actual concentrations is 13mg/ml, so the beta lactoglobulin concentration that this milk appearance is contained is 13g/L.
Claims (7)
1. detection method that detects beta-lactoglobulin content; It is characterized in that: comprise the steps: 1) beta lactoglobulin is dissolved in sample buffer; Prepare the standard solution of a plurality of different content beta lactoglobulins, and obtain the typical curve between beta-lactoglobulin content and the testing result after a plurality of solution are detected respectively; 2) sample is detected, and obtain testing result; 3), obtain the content of beta lactoglobulin in the sample with described testing result and the comparison of described typical curve; Wherein: the equipment of test sample is the high voltage capillary electrophoresis analyser;
Wherein, the use detected parameters that high voltage capillary electrophoresis is analyzed is following: the temperature that Capillary Electrophoresis detects is 15～40 ℃, capillary column diameter 25～75 μ m, length 20～600mm; DAD or UV-detector; Pressure sample introduction, pressure are 0～1psi, and sample injection time is 1s～20s; Separation temperature is 15～40 ℃, and WV is 7～30kV;
Electrophoretic is a zone electrophoresis during detection of high voltage capillary electrophoresis analysis, and its electrophoretic buffer is pH2.0～9.0, and composition is the inorganic salts damping fluid.
2. detection method according to claim 1; It is characterized in that: the method for the formulation of typical curve is following in the said step 1): preparation beta lactoglobulin standard solution; Dissolving beta lactoglobulin standard substance is mixed with and is respectively 2mg/ml, 1mg/ml, 0.5mg/ml, 0.25mg/ml, 0.1mg/ml in sample buffer; Prepare standard solution; Shake up the back and use the high voltage capillary electrophoresis analysis, obtain testing result, promptly obtain the relation curve of the beta-lactoglobulin content and the average correction factor; Wherein, described testing result is calculated according to following method: go out the average correction factor by calculated by peak area.
3. according to claim 1 or claim 2 detection method; It is characterized in that: pack standard solution or detected sample in the centrifuge tube into, centrifugal 5-15min under the 2000-6000r/min centrifugal condition, with sample and damping fluid according to 1: the ratio of 4-20 adds frozen pipe; And shake up; Accurately extract 10-200ul and handle in the sample adding sample hose, be positioned over supersonic wave cleaning machine 1～10min, use HPCE to measure.
4. detection method as claimed in claim 3 is characterized in that: described sample buffer is: the damping fluid that protein is disperseed is following damping fluid:
Add 40mmol/L 3-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%, pH value are 8.5
5. detection method as claimed in claim 2 is characterized in that: the described step that shakes up adopts eddy mixer.
6. detection method as claimed in claim 1 is characterized in that: described capillary column is a quartz capillary column, comprises coating and coating not.
7. according to the detection method described in the claim 2; It is characterized in that: the disposal route of described testing result is following: testing result is the collection of illustrative plates of sample separation; The utilization HPCE with analysis software, the area at each peak of integration calculates sample solution concentration; Take advantage of extension rate again, promptly get actual concentrations.
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| CN101477079A (en) * | 2009-01-06 | 2009-07-08 | 中国农业大学 | Active gel electrophoresis method for lactalbumin |
| CN101782549A (en) * | 2010-03-29 | 2010-07-21 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for detecting the content of casein phosphopeptide in milk products |
| CN104777257B (en) * | 2015-04-30 | 2017-01-18 | 澳优乳业（中国）有限公司 | Fast separation and detection method for whey protein components in dairy product |
| CN106526063A (en) * | 2016-11-09 | 2017-03-22 | 无锡艾科瑞思产品设计与研究有限公司 | Grain protein content detection method |
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| US5959171A (en) * | 1994-08-17 | 1999-09-28 | Pharming B.V. | Method for the production of biologically active polypeptides in a mammal's |
| CN1343779A (en) * | 2000-09-19 | 2002-04-10 | 上海博德基因开发有限公司 | Polypeptide-protein 12.21 containing peroxidase characteristics and polynucleoitde for coding it |
| CN1663961A (en) * | 2004-09-29 | 2005-09-07 | 天津商学院 | Technology for separating and purifying lactoferritin from cow colostrum |
| CN101117351A (en) * | 2007-04-30 | 2008-02-06 | 北京济普霖生物技术有限公司 | Method for purifying restructuring lactoferrin from transgenic cow's milk |
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Patent Citations (4)
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|---|---|---|---|---|
| US5959171A (en) * | 1994-08-17 | 1999-09-28 | Pharming B.V. | Method for the production of biologically active polypeptides in a mammal's |
| CN1343779A (en) * | 2000-09-19 | 2002-04-10 | 上海博德基因开发有限公司 | Polypeptide-protein 12.21 containing peroxidase characteristics and polynucleoitde for coding it |
| CN1663961A (en) * | 2004-09-29 | 2005-09-07 | 天津商学院 | Technology for separating and purifying lactoferritin from cow colostrum |
| CN101117351A (en) * | 2007-04-30 | 2008-02-06 | 北京济普霖生物技术有限公司 | Method for purifying restructuring lactoferrin from transgenic cow's milk |
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