Establishment Of RP-HPLC Fingerprint Of Protein In Dairy Products And Its Application In Milk Source Analysis And Quality Control

The protein in milk is one of the estimated indicators for milk quality,and is related to the nutritional value of milk.It is important to establish a reasonable and stable method to evaluate milk proteins.In this paper,milk protein was analyzed by reversed phase high performance liquid chromatography and fingerprint technique.This method was used to detect the proteins from different source,thus to distinguish the milk samples.Firstly,a reversed phase chromatographic approach was established to separate and determine milk protein components.The influence factors of milk protein RP-HPLC separation,such as column temperature,flow rate,gradient and wavelength,were investigated;the conditions of separating milk protein by RP-HPLC were selected.The repeatability and precision of the method were verified;the repeatability and intermediate precision showed the RSD%of peak retention time were between 0.04%?1.36%and 0.38?1.33%,respectively;and RSD%of the peak area were between 5.22%?15.04%and 8.27%?30.73%,respectively.The standard of ?-CN,?-CN,?-CN,?-la and ?-1gB were used to fit the linear curves,respectively;the R2 were between 0.990?0.999.The bovine milk proteins were determined by the RP-HPLC retention time compared with the five milk protein standards,namely,?-CN,?-CN,?-CN,?-la and ?-lgB.Thus,the approach established the foundation for determination of proteins in milk samples.Secondly,the protein from different sources of bovine,sheep and goat's milk was analyzed,respectively.The protein of bovine milk was analyzed by RP-HPLC,and five major proteins were determined by standards.The ewe's milk protein was analyzed by RP-HPLC to compare the milk protein components difference in different sources.The molecular weight and content of each protein in bovine and ewe's milk were compared by SDS-PAGE,and the content of total protein from different milk was determined by BCA method.Finally,the fingerprints of protein in bovine and ewe's milk were established,respectively.The similarities of protein were analyzed by fingerprints.In six batches of bovine milk,the similaritis were between 0.981?0.998;in the seven batches of goat's milk,the similaritis were between 0.721?0.989;in the two batches of sheep's milk the similarity was 0.996;in the adulterate milk samples,the similarities between adulterate milk,bovine,sheep and goat's milk were 0.578?0.887.Principal component analysis was used to analyze different sources of protein in milk samples.Milk samples from bovine,sheep and goat belong to three categories;in the same categories,different sources of protein had certain dispersion between each sample point.In the adulterate milk samples,categories of adulterate sample points were away from the distribution of bovine and ewe's milk samples,which can identify the adulteration of dairy products.In summary,this paper established a stable RP-HPLC method to separate milk protein from different sources,and obtained protein chromatographic profiles of different milk samples;individual characteristics and whole characteristics of proteins in each milk were illustrated through the RP-HPLC profiles;through protein chromatographic fingerprint technology,similarity and difference were evaluated in different samples;thus fingerprint of certain milk can be established.The approach provided a reasonable technique to determine protein in milk,and provided a theoretical basis to evaluate milk quality and identify milk sources,and laid the foundation to establish the milk fingerprint of specific brand.