Study On The Detection Of Main Proteins By HPLC And UPLC-Q Exactive In Dairy Products

Dairy proteins include casein,whey protein and a little milk fat globular membrane protein.Casein(CN)contains four variants:?sl-CN,?s2-CN,?P-CN,?-CN.The content of the casein is 80%of the total milk protein and relatively constant,usually the content of casein in daily products reflects the level of its nutritional value;Immunoglobulin G(IgG)in whey protein as immune supplements in infants' formula,it is the main force not only to resist diseases for infants and young children,but also to improve their immunity.The level of IgG in infant formula milk powder directly determine the product pricing in the highly competitive market;The phenomenon of adulteration about bovine and goat milk has attracted a lot of attention in recent years,although the related detection technology has been increasing and maturing,there is relatively lacking about the corresponding screening identification technology based on the difference between their peptides.Therefore,it is of great significance in monitoring the value of dairy products and food safety to develop an optimized method for separation of casein by HPLC,to establish a method for determining the content of immunoglobulin G and to establish a detection technology to identify adulteration about bovine and goat milk based on the peptides.The paper make a study on the chromatographic separation conditions of casein,the content of immunoglobulin G in infants' formula and the determination a identify technology based on specific peptides of bovine and goat milk by using reversed phase high performance liquid chromatography(RP-HPLC),high performance gel permeation chromatographic(HPGPC)and ultra-high performance liquid chromatography-quad column/electrostatic orbitrap high resolution mass spectrometry(UPLC-Q Enactive)technology.Including the following contents:1.An optimization method by reversed phase high performance liquid chromatography was developed for the separation of casein.Samples were mixed and incubated with a buffer system consisting of urea,trimethyl aminomethane,sodium citrate and ?-mercaptoethanol.The separation of casein conditions was optimized by comparison with standard chromatographic peak retention time.Results showed that the separation was the best when the extraction solution was a buffer system consisting of urea,trimethyl aminomethane.sodium citrate and ?-mercaptoethanol,the detection wavelength was 220 nm,the chromatographic column was C4 column(4.6x250mm,5 ?m),while the optimization of the mobile phase gradient also reduced the experimental time.The method is simple and reliable,and is suitable for the qualitative of the separation of casein by RP-HPLC.2.A high performance gel permeation chromatographic detection method was developed for the determination of Immunoglobulin G in formula milk powder.Samples were dissolved with pH 7,0.05 mol/L disodium hydrogen phosphate buffer;then were removed fat by high-speed centrifugation and filtration.The filtered liquor was purified by immunoaffinity column,monitored by the HPGPC,and quantified by the matrix-matched calibration curves.Results showed that good linarites in the range of 0.2-5.0 mg/mL,with the correlation coefficients(r)higher than 0.9990,the recoveries were ranged from 87.2%to 117.7%with relative standard deviation between3.72%and 7.09%.The limit of detection methodwaslOmg/100g.The method has the advantages of convenience,accuracy and is suitable for the qualitative and quantitative determination of IgG in formula milk powder.3.The samples were extracted by water and digested with trypsin,the determination was carried by UPLC-Q Exactive,Results showed that there are three peptides in the milk:880.475?1157.633?1557.359,two peptides in the goat:990.036?931.523 peptides and four common peptides:837.475?851.097?1094.087?1217.027 at a mass ratio of 200-2000.4.A screening and quantitative method for adulteration about bovine and goat milk based on their specific peptides was developed by the UPLC-Q Exactive technique,then quantified by the ratio of bovine and goat milk of matrix matched external standard method.There is good linarites in the high ratio of 0%?100%and the low ratio of 0%-10%in bovine,with the correlation coefficients(r)higher than 0.9990.Compared with theoretical values,the recoveries were ranged from 96.97%to 112.4%with relative standard deviation between3.77 and 14.72%.The limit of detection method wasl%.The method and can be used for the screening and quantification of adulteration about bovine and goat milk.